AU748639B2 - Receptors for TGF-beta-related neurotrophic factors - Google Patents

Description

WO 98/46622 PCT/US98/07996 RECEPTORS FOR TGF-B-RELATED NEUROTROPHIC FACTORS Reference to Government Grants This invention was made with government support under National Institutes of Health Grant Numbers R01 AG13729 and R01 AG13730. The government has certain rights in this invention.

Related Applications This application claims the benefit of U.S.

Provisional Application entitled TrnR2, A Novel Receptor Which Mediates Neurturin And GDNF Signaling Through Ret filed April 17, 1997.

Background of the Invention Field of the Invention This invention relates generally to receptors for trophic or growth factors and, more particularly, to a novel receptor for TGF-p-related neurotrophic factors.

Description of the Related Art The development and maintenance of tissues in complex organisms requires precise control over the processes of cell proliferation, differentiation, survival and function. A major mechanism whereby these processes are controlled is through the actions of polypeptides known as "growth factors". These structurally diverse molecules act through specific cell surface receptors to produce these actions.

Of particular importance are those growth factors, termed "neurotrophic factors", that promote the differentiation, growth and survival of neurons and reside in the nervous system or in innervated tissues.

Nerve growth factor (NGF) was the first neurotrophic factor to be identified and characterized (Levi- Montalcini et al., J. Exp. Zool. 116:321, 1951 which is WO 98/46622 PCT/US98/07996 2 incorporated by reference). NGF exists as a noncovalently bound homodimer that promotes the survival and growth of sympathetic, neural crest-derived sensory, and basal forebrain cholinergic neurons.

In recent years it has become apparent that growth factors fall into classes, i.e. families or superfamilies based upon the similarities in their amino acid sequences. Examples of such families that have been identified include the fibroblast growth factor family, the neurotrophin family and the transforming growth factor-beta (TGF-B) family. As an example of family member sequence similarities, TGF-p family members have 7 canonical framework cysteine residues which identify members of this superfamily.

NGF is the prototype member of the neurotrophin family. Brain-derived neurotrophic factor (BDNF), the second member of this family to be discovered, was shown to be related to NGF by virtue of the conservation of all six cysteines that form the three internal disulfides of the NGF monomer (Barde, Prog Growth Factor Res 2:237-248, 1990 and Liebrock et al. Nature 341:149-152, 1989 which are incorporated by reference). By utilizing the information provided by BDNF of the highly conserved portions of two factors, additional members (NT-3, NT- 4/5) of this neurotrophin family were rapidly found by several groups (Klein, FASEB J 8:738-44, 1994 which is incorporated by reference).

Signal transduction for neurotrophins is mediated by a family of closely related tyrosine kinase receptors (Trk). The known members of the Trk family of receptors are: TrkA, identified as the NGF receptor; TrkB, which mediates signaling by BDNF and NT-4/5; and TrkC, which transduces the signals of NT-3 (for review, see (Tuszynski et al., Ann Neurol 35:S9-S12, 1994 which is incorporated by reference). In addition to these preferential specificities, there is evidence of cross- WO 98/46622 PCT/US98/07996 3 talk between different members of the neurotrophin and Trk families, particularly in fibroblast-based model systems. For example, NT-3 stimulates phosphorylation of TrkB expressed in fibroblasts with a dose-response relationship equivalent to that of BDNF and NT-4/5 (Ip et al, Neuron 10:137-149, 1993, incorporated herein by reference), while in PC12 cells, the TrkB receptor is 100-fold more sensitive to stimulation by BDNF and than by NT-3. NT-3 may also signal through TrkA, although with different specificities than NGF (Ip et al, supra and Cordon-Cardo et al, Cell 66:173-183, 1991, incorporated herein by reference).

Recently, a new family of neurotrophic factors has been identified whose members are not structurally related to NGF and other neurotrophins but are structurally similar to TGF-p. As described in copending applications 08/519,777 and 08/615,944, which are incorporated herein by reference, the known members of this new family, which has been named TRN (TGF-p Related Neurotrophic factors), are glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), and persephin (PSP).

The placement of human GDNF and NTN into the same growth factor family is based on the similarities of their physical structures and biological activities.

These two proteins have 42% identity in their amino acid sequences including seven cysteine residues whose positions are exactly conserved in neurturin and GDNF.

The biological activities of GDNF and NTN include supporting the survival of rat superior cervical, nodose, and dorsal root ganglion neurons in vitro, although NTN is more potent than GDNF in promoting SCG survival (Kotzbauer et al., supra). In addition, as disclosed in the copending international patent application, WO 97/08196, which is incorporated herein by reference, the accumulation of radiolabeled NTN in the sensory neurons WO 98/46622 PCT/US98/07996 4 following injection into partially crushed sciatic nerves of rats can be blocked by a 100 fold excess of unlabeled NTN or unlabeled GDNF, suggesting that NTN and GDNF compete for the same receptor.

Recently, it was reported that GDNF acts through a multicomponent receptor complex in which a transmembrane signal transducing component, the Ret protein-tyrosine kinase (Ret or Ret PTK), is activated upon the binding of GDNF with another protein, called GDNF Receptor a (GDNFRa) which has no transmembrane domain and is attached to the cell surface via a glycosyl-phosphatidylinositol (GPI) linkage (Durbec et al., Nature 381:789-793, 1996; Jing et al., Cell 85:1113-1124, 1996; Treanor et al., Nature 382:80-83, 1996; Trupp et al., Nature 381:785-789, 1996, which are incorporated herein by reference).

GDNF

also induces activation of the Ret PTK when a soluble form of GDNFR-a is added to the culture medium along with GDNF, demonstrating that GDNFR-a does not need to be anchored to the cell membrane to interact with Ret (Jing et al., supra). The formation of a functional

GDNF

receptor complex by GDNFR-a and Ret is supported, in part, by the observations that mice deficient in either GDNF or Ret are phenotypically similar and that GDNFR-a and Ret are expressed together in the developing nephron, midbrain, and motor neurons, all known targets of GDNF action.

Neuronal degeneration and death occur during development, during senescence, and as a consequence of pathological events throughout life. It is now generally believed that neurotrophic factors regulate many aspects of neuronal function, including survival and development in fetal life, and structural integrity and plasticity in adulthood. Since both acute nervous system injuries as well as chronic neurodegenerative diseases are characterized by structural damage and, possibly, by disease-induced apoptosis, it is likely that neurotrophic WO 98/46622 PCT/US98/07996 factors play some role in these afflictions. Indeed, a considerable body of evidence suggests that neurotrophic factors may be valuable therapeutic agents for treatment of these neurodegenerative conditions, which are perhaps the most socially and economically destructive diseases now afflicting our society. Nevertheless, because different neurotrophic factors can act preferentially through different receptors and on different neuronal cell types, there remains a continuing need for the identification and characterization of these receptors of growth factors in the diagnosis and treatment of a variety of acute and chronic diseases of the nervous system.

Summary of the Invention: Briefly, therefore, the present invention i-s directed to the identification and isolation of substantially purified polypeptides that mediate the survival and growth promoting effects of neurotrophic factors on neurons. Accordingly, the inventors herein have succeeded in discovering that members of the TRN growth factor family share receptors and signal transduction pathways. In particular, the inventors have discovered that signaling of NTN and GDNF through the Ret tyrosine kinase receptor is mediated by a novel family of co-receptors, referenced herein as TrnR (TGF-p-related neurotrophic factor Receptors). The TrnR co-receptor family includes the known co-receptor protein GDNFR-a, referred to herein as TrnRl, and a novel protein, TrnR2, either of which can form a functional receptor complex with Ret for both NTN and GDNF.

The existence of this co-receptor family was established by the isolation and characterization of a cDNA encoding TrnR2 which shows significant homology with TrnRl. In particular, a comparison of their respective predicted amino acid sequences revealed that human TrnRl and human TrnR2 are 48% identical at the amino acid level WO 98/46622 PCT/US98/07996 6 and share 30 of 31 cysteine residues with nearly identical spacing, indicating a conserved cysteine backbone structure. Both co-receptors also contain a predicted N-terminal signal sequence, a putative Cterminal GPI linkage signal peptide (GPIsp), and three potential N-linked glycosylation sites.

Recently, new nomenclature for this family of GPIlinked co-receptors for GDNF and neurturin has been adopted by scientists in the field such that the official name for the TrnR family is now GFRa, with individual members of the family being named GFRal (previously known as GDNFRa, TrnRl and RetL1), GFRa2 (previously TrnR2, NTNRa and RetL2) and GFRa3 (previously TrnR3).

Nomenclature Committee, Neuron 19(3):485, 1997. However, the older TrnR nomenclature will be used herein.

Accordingly, the invention provides a substantially purified TrnR2 polypeptide. It is believed that TrnR2 homologs of different mammalian species have at least 85% amino acid sequence identity while amino acid sequence identity may be as low as 65% in TrnR2 homologs of non-mammalian species such as avian species.

TrnR2 polypeptides identified herein include predicted precursor and mature forms of TrnR2 protein in which the predicted mature protein lacks the N-terminal signal sequence and the C-terminal GPIsp but is otherwise identical to the precursor protein. Human precursor and mature proteins have the predicted amino acid sequences set forth in SEQ ID NOS:2 and 3, respectively. The corresponding predicted precursor and mature forms of mouse TrnR2 protein have the amino acid sequences shown in SEQ ID NOS:5 and 6 (Figure In addition, TrnR2 polypeptides of the invention include variants of human and mouse precursor proteins translated from alternatively spliced TrnR2 mRNA having the amino acid sequences shown in SEQ ID NOS:7 and 8. Soluble TrnR2 polypeptides which lack a GPI anchor are also

(I

WO 98/46622 PCT/US98/07996 7 contemplated by the invention. Such soluble TrnR2 polypeptides include soluble forms of alternatively spliced variants of TrnR2. TrnR2 polypeptides also include biologically active fragments of the full-length precursor or mature proteins which are capable of binding a TRN growth factor, or which are capable of activating the Ret PTK in the presence of the TRN growth factor, or which are capable of eliciting in a host animal antibodies specific for TrnR2.

The present invention also provides nucleotide sequences that encode a TrnR2 polypeptide. Human precursor and mature TrnR2 proteins are encoded by residues 36 to 1427 and residues 99 to 1331, respectively, of the nucleotide sequence set forth in SEQ ID NO:1. Mouse precursor and mature TrnR2 proteins are encoded by residues 1 to 1389 and residues 64 to 1296, respectively, of the nucleotide sequence set forth in SEQ ID NO:4.

Expression vectors and stably transformed cells are also provided. The transformed cells can be used in a method for producing a TrnR2 polypeptide.

In another embodiment, the present invention provides a method for preventing or treating neuronal degeneration comprising administering to a patient in need thereof a therapeutically effective amount of a TrnR2 polypeptide, optionally along with a therapeutically effective amount of NTN or GDNF. A patient may also be treated by implanting transformed cells which express a TrnR2 polypeptide or a DNA sequence which encodes TrnR2 into a patient's tissues which would benefit from increased sensitivity to a TRN such as NTN or GDNF. In another embodiment, a patient with neuronal degeneration is treated by implanting neuronal cells cultured and expanded by growth in the presence of TrnR2 and NTN or GDNF.

WO 98/46622 PCT/US98/07996 8 Another embodiment provides a method for treating tumor cells by administering a composition comprising an effective amount of TrnR2 and an effective amount of NTN or GDNF or a composition comprising DNA sequences encoding TrnR2 and NTN or GDNF to produce a maturation and differentiation of the cells.

Yet another embodiment involves the use of a soluble TrnR2 polypeptide as an agonist of TRN growth factors.

In another embodiment the present invention provides isolated and purified TrnR2 antisense polynucleotides.

The present invention also provides compositions and methods for detecting TrnR2 expression. One method detects TrnR2 protein using anti-TrnR2 antibodies and other methods are based upon detecting TrnR2 mRNA using recombinant DNA techniques.

Among the several advantages found to be achieved by the present invention, therefore, may be noted the provision of a new co-receptor for neurturin and GDNF which mediates the ability of these growth factors to maintain and prevent the atrophy, degeneration or death of certain cells, in particular neurons; the provision of other members of the TrnR family of growth factor receptors by making available new methods capable of obtaining said other family members; the provision of methods for obtaining TrnR2 by recombinant techniques; the provision of methods for preventing or treating diseases producing cellular degeneration and, particularly, neuronal degeneration; the provision of methods for limiting the effects of TRN growth factors in a patient; and the provision of methods that can detect and monitor TrnR2 levels in a patient.

WO 98/46622 PCT/US98/07996 9 Brief Description of the Drawings Figure 1 illustrates the homology of the amino acid sequences for the predicted precursor forms of TrnRl (human, SEQ ID NO:12; rat, SEQ ID NO:13) and TrnR2 (human, SEQ ID NO:2; mouse, SEQ ID NO:5) with identical amino acid residues enclosed in boxes and shared cysteine residues shaded; Figure 2 illustrates the nucleotide sequence (SEQ ID NO:4) and amino acid translation (SEQ ID NO:5) of the long splice variant of precursor mouse TrnR2 with the predicted N-terminal signal sequence and C-terminal hydrophobic domain underlined, a potential GPI attachment site indicated by a asterisk, the potential N-linked glycosylation sites enclosed in boxes, and the amino acid region missing in the short splice variant shaded; Figures 3A-C illustrate the effect of NTN and GDNF on Ret phosphorylation as detected by an immunoassay using antibodies specific for phosphotyrosine and Ret in (Fig. 3A) fibroblasts stably transfected with Ret alone (Ret) or both Ret and the long splice variant of TrnR2 (Ret/TrnR2) and treated with GDNF or NTN or not treated (Fig. 3B) fibroblasts expressing both Ret and TrnR2- LV which were pre-treated or not treated with phosphatidylinositol-specific phospholipase C (PIPLC) before growth factor treatment, and (Fig. 3C) fibroblasts stably expressing both Ret and TrnR2-LV (TrnR2/Ret) or Ret and TrnRl (TrnRl/Ret) and treated with increasing amounts of NTN or GDNF; Figure 3D illustrates the effect of GDNF, NTN, and persephin (PSP) on Ret tyrosine phosphorylation as detected by an immunoassay using antibodies specific for phosphotyrosine and Ret in fibroblasts coexpressing Ret and either the long splice variant of TrnR2 (TrnR2-LV) or the short splice variant (TrnR2-SV); Figure 3E illustrates the binding affinities of soluble TrnR2-LV fused with the Fc region of human IgG, WO 98/46622 PCT/US98/07996 (R2-Ig) for GDNF, NTN and PSP as measured in an ELISA binding assay; Figure 4 illustrates the tissue distribution of TrnR2 mRNA in adult mouse showing a Northern blot of total RNA probed with a 32 P-labeled TrnR2 cDNA fragment; Figures 5A-D illustrates the expression of TrnRl, TrnR2, and Ret in known sites of GDNF and/or NTN action showing in situ hybridization analysis using 33 P-labeled RNA probes of tissue samples from (Fig. 5A) E14 mouse (developing) ventral mesencephalon (Fig. 5B) adult mouse spinal cord, (Fig. 5C) E14 mouse (developing) kidney gut and dorsal root ganglia (drg), and (Fig. 5D) adult rat superior cervical ganglion (SCG); Figure 6 illustrates TrnRl, TrnR2, and Ret expression in primary SCG cultures containing a contaminating population of non-neuronal cells showing the amount of different mRNAs at varying times after removal of nerve growth factor as measured by reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for Ret, TrnR2, neuron-specific enolase NSE, TrnRl, and a Schwann cell marker (S100); Figure 7 illustrates that expression of TrnR1, but not TrnR2, is up-regulated in the distal sciatic nerve after nerve injury as shown by Northern blot analysis of total RNA isolated from normal sciatic nerve and the distal segment of sciatic nerve seven days posttransection (7D) using 32 P-labeled TrnRl and TrnR2 probes and brain RNA as a positive control for the detection of TrnR2 mRNA.

Figure 8 illustrates the expression of GF (TRN) receptors and neurturin in the adult mouse forebrain showing darkfield photographs of coronal sections analyzed by in situ hybridization using 33 P-labeled riboprobes to detect expression of GFRa-1 (TrnR1) (Fig.

8A), GFRa-2 (TrnR2) (Fig. 8B), Ret (Fig. 8C) and NTN (Fig. 8D), in which the various regions are abbreviated WO 98/46622 PCT/US98/07996 11 as Cg-cingulate cortex, Cl-claustrum, DBB-nucleus of the diagonal band of Broca, DEn-dorsal endopiriform nucleus, LS-lateral septal nucleus, MS-medial septal nucleus, Pir-piriform cortex, Tu-olfactory tubercle, and VP-ventral pallidum; Figure 9 illustrates the expression of GF (TRN) receptors and neurturin in in the neocortex, hippocampus, thalamus, and hypothalamus showing darkfield photographs of coronal sections of the adult mouse brain analyzed by in situ hybridization using 33 P-labeled riboprobes to detect expression of (Fig. 8) GFRa-1 (TrnRl), (Fig. 8B) GFRa-2 (TrnR2), (Fig. 8C) Ret, and (Fig. 8D) NTN, in which the various regions are abbreviated as Th-thalamic nuclei, A-amygdala, H-hypothalamus, LD-laterodorsal nucleus of the thalamus, MD-mediodorsal nucleus of the thalamus, MHb-medial habenula, Rt--reticular thalamic nucleus, STh-subthalamic nucleus, and ZI--ona incerta; Figure 10 illustrates the expression of GF (TRN) receptor components in the adult mouse midbrain showing darkfield photographs of coronal sections of adult mouse midbrain analyzed by in situ hybridization using 33

P-

labeled riboprobes to detect expression of (Fig. GFRa-1 (TrnRl) in the compacta region of the substantia nigra, the VTA, the oculomotor nucleus and the superficial layers of the superior colliculus, (Fig. GFRa-2 in the compacta region of the substantia nigra, in the VTA, and the oculomotor nucleus, (Fig. 10C) Ret mRNA in the SNc and the VTA, in which the various regions are abbreviated as 3-oculomotor nucleus, SN-substantia nigra, SuMM-supramammillary nucleus, MGN-medial geniculate nucleus, and VTA-ventral tegmental area; Figure 11 illustrates the expression of NTN mRNA in the supraoptic and paraventricular nuclei of the hypothalamus showing (Fig. 11A) a darkfield photograph and (Fig. 11B-11C) brightfield photographs at higher magnification which show detection of NTN expression in WO 98/46622 PCT/US98/07996 12 magnocellular neurons in the supraoptic (Fig. 11B) and paraventricular (Fig. 11C) nuclei with the various regions abbreviated as PV-paraventricular nucleus, SO-supraoptic nucleus, and 3V-third ventricle; Figure 12 illustrates expression of GDNF and GF (TRN) receptor mRNA in adult mouse midbrain and brainstem showing darkfield photographs of coronal sections analyzed by in situ hybridization using "P-labeled riboprobes to detect expression of (Fig. 12A) Ret mRNA in cranial nerve nuclei 10 and 12, and in the gigantocellular reticular nucleus (Fig. 12B) GFRa-2 (TrnR2) in cranial nerve nuclei Sp5, 6, 7 and ventral cochlear nucleus (Fig. 12C) GDNF in the VC and the facial motor nucleus, (Fig. 12D) GFRa-1 in the facial motor nucleus and in the dorsal cochlear nucleus (DC), (Fig. 12E) GFRa-2 in the inferior colliculus the tegmental nuclei, and the locus coeruleus, (Fig. 12F) Ret mRNA in the trigeminal motor nucleus and the inferior colliculus, with the various regions being identified as 6-abducens nucleus, 7-facial nucleus, 10-vagal motor nucleus, 12-hypoglossal nucleus, Gi-gigantocellular reticular nucleus, IC-inferior colliculus, LG-locus coeruleus, Sp5-spinal trigeminal nucleus, trigeminal nucleus, Tg-tegmental nuclei, VG-ventral cochlear nucleus; Figure 13 illustrates GF (TRN) receptor expression in adult mouse cervical spinal cord showing darkfield photographs of transverse sections analyzed by in situ hybridization using 33 P-labeled riboprobes to detect expression of (Fig. 13A) GFRa-1 (TrnRl), (Fig. 13B) GFRa- 2 (TrnR2), and (Fig. 13C) Ret, with various regions identified as DH-dorsal horn, and VH-ventral horn; Figure 14 illustrates GF (TRN) receptor and NTN mRNA expression in adult cerebellum showing darkfield photographs of sagittal sections analyzed by in situ hybridization using 33 P-labeled riboprobes to detect WO 98/46622 PCT/US98/07996 13 expression of (Fig. 14A) GFRa-1 (TrnRl) in cells adjacent to Purkinje neurons in the Purkinje layer, (Fig. 14B) GFRa-2 (TrnR2) in the granule cell layer and in neurons that appear to be Purkinje cells in the Purkinje layer, (Fig. 14C) Ret in the Purkinje layer in cells surrounding Purkinje neurons and in the molecular layer, and (Fig.

14D) NTN in the Purkinje and granule cell layers, with the various regions abbreviated as Gr-granule cell layer, P-Purkinje layer, Mol-molecular layer.

Description of the Preferred Embodiments The present invention is based upon the surprising discovery that NTN, like GDNF, can stimulate Ret PTK through the known co-receptor GDNFR-a and thereby cause the activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI-3-K) intracellular signaling pathways. As the first coreceptor known to mediate signaling by at least two members of the TRN family of growth factors, GDNFR-a is referred to herein as TrnRl. The unexpected discovery that members of the TRN family of growth factors share a receptor complex and signal transduction pathways led to the identification, isolation and sequencing of a cDNA encoding a novel second co-receptor for NTN and GDNF, TrnR2. Prior to this invention, TrnR2 was unknown and had not been identified as a discrete biologically active substance, nor had it been isolated in pure form.

TrnR2 was identified by searching a database of Expressed Sequence Tags (dbEST database) using the Basic local alignment search tool (BLAST, Altschul et al., J.Mol.Biol. 215:403-410, 1990 incorporated herein by reference) and the full length rat TrnRl protein sequence (GenBank Accession No. U59486) as a query. Three human ESTs (H12981, R02135, W73681) which showed only partial, but significant, homology to rat TrnRl were identified by the BLAST search. The determination and alignment of the WO 98/46622 PCT/US98/07996 14 complete sequences of these three ESTs, obtained from the WashU-Merck EST project, indicated that they all encoded partial cDNAs of an identical transcript.

The 5' end of the cDNA was obtained by the rapid amplification of cDNA ends (RACE) technique using as templates human brain and placenta cDNA libraries (Marathon RACE libraries, Clontech, Palo Alto, CA) and the Klentaq LA polymerase chain reaction (PCR) technique described by Barnes, Proc.Natl.Acad.Sci.U.S.A. 91:2216- 2220, 1994, incorporated herein by reference. Two alternatively spliced forms of TrnR2 mRNA were identified in both brain and placenta, the short splice variant (TrnR2-SV) is missing 399 nucleotides of the coding sequence from the 5' end of the long splice variant (TrnR2-LV) (residues 75 to 473 of SEQ ID NO:1). The predicted amino acid sequence of the long splice variant of human precursor TrnR2 contains 464 amino acids and is shown in SEQ ID NO:2, the predicted amino acid sequence for the mature protein is shown in SEQ ID NO:3. The short splice variant has a predicted amino acid sequence of 331 amino acids as set forth in SEQ ID NO:7, which would be encoded by nucleotides 36-74 and 474-1427 of SEQ ID NO:1. The corresponding mouse cDNAs for both the long and short splice variants were also obtained by PCR, using a brain cDNA template. The full length precursor murine cDNA is set forth in SEQ ID NO:4 and contains a single long open reading frame (ORF) encoding a predicted protein of 463 amino acids (SEQ ID NO:5); the short splice variant identified has an ORF encoding a predicted 330 amino acid polypeptide (SEQ ID NO:8) which would be encoded by nucleotides 1-39 and 438-1389 of SEQ ID NO:4.

All physical features of TrnR2 indicate that it is closely related to TrnRl. As shown in Figure 1, the predicted amino acid sequence for TrnR2-LV shows significant homology with TrnRl. (The human and rat TrnRl sequences (SEQ ID Nos. 12 and 13, respectively) are those WO 98/46622 PCT/US98/07996 reported by Jing et al., supra.) The predicted protein for precursor TrnR2 contains a putative 21 amino acid signal sequence (residues 1-21 of SEQ ID NO:2 and SEQ ID for human and mouse proteins, respectively) at the amino terminus, three potential N-linked glycosylation sites, and has a stretch of 16 carboxyl-terminal hydrophobic amino acids (residues 449-464 of SEQ ID NO:2 and 448-463 of SEQ ID NO:5 for human and mouse proteins, respectively). The presence of the N- and C-terminal hydrophobic regions indicates that mature TrnR2 is potentially a GPI-linked protein (Udenfriend and Kodukula, Ann. Rev. Biochem. 64:563-591, 1995, incorporated herein by reference), as has been demonstrated for TrnRl (Treanor et al. supra; Jing et al., supra). A potential GPI attachment site for the human and mouse long splice variants is the glycine residue at position 411 of SEQ ID NO:2 and 3, respectively. Accordingly, the predicted GPI attachment site in the short splice variant is the glycine residue at position 299 of SEQ ID NO:7 for the human protein and at position 299 of SEQ ID NO:8 for the mouse protein.

The inventors herein have found significant functional similarities and dissimilarities between TrnR2 and TrnRl. Experimental data which is discussed below indicate that either NTN or GDNF can activate Ret in the presence of either TrnR2 or TrnRl. In addition, Ret activation in the presence of either co-receptor responds to stimulation with either NTN or GDNF in a dosedependent manner. However, while the TrnRl/Ret receptor complex responds equivalently to the same amounts of each ligand, the TrnR2/Ret complex is more sensitive to NTN than GDNF, indicating that, at least in the model system examined, the latter complex may function preferentially as a NTN receptor. Also, TrnRl and TrnR2 are expressed in a partially overlapping manner. While both coreceptors are expressed in the dorsal root ganglia (DRG) WO 98/46622 PCTIUS98/07996 16 and the brain, TrnR2 is not expressed or expressed at very low levels in several known targets of GDNF action in which both TrnRl and Ret are expressed, including embryonic and adult nigra, motor neurons, gut and kidney.

In contrast, TrnR2 and Ret appear to comprise the expressed receptor complex in SCG neurons.

Although the data indicate that a physiological pairing of ligand and receptor may exist for the TrnR family as it does for the Trk family, there is also in vivo evidence of cross-talk between the TRN ligands and their receptors as observed in several ligand-receptor systems having multiple family members, including between members of the neurotrophin growth factor and Trk receptor families. For example, the inventors herein have found that neuroblastoma cell lines may express either TrnRl, TrnR2 or both, but despite this heterogeneity, those cell lines which respond to GDNF or NTN always respond to both factors. Thus, it is believed that TrnR2, TrnRl or other as yet unidentified members of the TrnR family can combine in vivo with Ret to form a functional receptor complex for NTN and GDNF, and possibly for persephin and other as yet unidentified members of the TRN growth factor family as well.

Accordingly, the invention provides a substantially purified TrnR2 polypeptide. A TrnR2 polypeptide of the invention includes growth factor receptors of any origin which are substantially homologous to and which are biologically equivalent to the human or mouse TrnR2 polypeptides characterized and described herein. Such substantially homologous growth factor receptors may be native to any tissue or species and, similarly, biological activity can be characterized in any of a number of biological assay systems.

The term "biologically equivalent" is intended to mean that the compositions of the present invention are capable of demonstrating some or all of the same signal WO 98/46622 PCT/US98/07996 17 mediating properties in a similar fashion, not necessarily to the same degree, as the recombinantly produced human or mouse TrnR2.

By "substantially homologous" it is meant that the degree of amino acid homology of human or mouse TrnR2 to a TrnR2 from any species is greater than that between TrnR2 and TrnRl (GDNFR-a).

Sequence identity or percent identity is intended to mean the percentage of identical residues between two sequences, referenced to human TrnR2 when determining percent identity with non-human TrnR2, referenced to TrnR2 when determining percent identity with non-TrnR2 growth factor receptors and referenced to human TrnR1 when determining percent identity of non-TrnR2 growth factor receptors with TrnRl, when-the two sequences are aligned using the Clustlal method (Higgins et al, Cabios 8:189-191, 1992) of multiple sequence alignment in the Lasergene biocomputing software (DNASTAR, INC, Madison, WI). In this method, multiple alignments are carried out in a progressive manner, in which larger and larger alignment groups are assembled using similarity scores calculated from a series of pairwise alignments. Optimal sequence alignments are obtained by finding the maximum alignment score, which is the average of all scores between the separate residues in the alignment, determined from a residue weight table representing the probability of a given amino acid change occurring in two related proteins over a given evolutionary interval.

Penalties for opening and lengthening gaps in the alignment contribute to the score. The default parameters used with this program are as follows: gap penalty for multiple alignment 10; gap length penalty for multiple alignment 10; k-tuple value in pairwise alignment 1; gap penalty in pairwise alignment 3; window value in pairwise alignment 5; diagonals saved in pairwise alignment 5. The residue weight table used WO 98/46622 PCT/US98/07996 18 for the alignment program is PAM250 (Dayhoff et al., in Atlas of Protein Sequence and Structure, Dayhoff, Ed., NBRF, Washington, Vol. 5, suppl. 3, p. 345, 1978).

Percent conservation is calculated from the above alignment by adding the percentage of identical residues to the percentage of positions at which the two residues represent a conservative substitution (defined as having a log odds value of greater than or equal to 0.3 in the PAM250 residue weight table). Conservation is referenced to human TrnR2 when determining percent conservation with non-human TrnR2, and referenced to TrnR2 when determining percent conservation with non-TrnR2 growth factor receptors. Conservative amino acid changes satisfying this requirement are: R-K; E-D, Y-F, L-M; V-I, Q-H. The calculations of identity and conservation between human and mouse TrnR2 (hTrnR2 and mTrnR2, respectively) and between each of these and human and rat TrnRl (hTrnRl and rTrnRl, respectively) are shown in Table 1.

Table 1 COMPARISON IDENTITY CONSERVATION hTrnR2 v. mTrnR2 94 hTrnR2 v. hTrnRl 48 53 hTrnR2 v. rTrnRl 47 52 mTrnR2 v. hTrnRl 43 47 mTrnR2 v. rTrnRl 47 52 The degree of homology between the predicted precursor mouse and human TrnR2 proteins is about 94% sequence identity and all TrnR2 homologs of non-human mammalian species are believed to have at least about sequence identity with human TrnR2. For non-mammalian species such as avian species, it is believed that the degree of homology with TrnR2 is at least about identity. By way of comparison, the variations between members of the TrnR family of receptors can be seen by comparing TrnRl and TrnR2 (Fig. Human and mouse precursor TrnR2 share about 94% identical amino acids and WO 98/46622 PCT/US98/07996 19 have about 53% and 52% sequence conservation with human and rat precursor TrnRl, respectively. It is believed that the different TrnR family members similarly have a sequence identity of about 40% to that of TrnR2 and about 40% to that of TrnRl and within a range of about 30% to about 85% identity with TrnR2 and within a range of about to about 85% sequence identity with TrnRl. Thus, a given non-TrnR2 and non-TrnRl family member from one species would be expected to show lesser sequence identity with TrnR2 and with TrnRl from the same species than the sequence identity between human TrnR2 and TrnR2 from a non-human mammalian species, but greater sequence identity than that between human TrnR2 and any other known growth factor receptor except TrnRl.

A TrnR2 polypeptide of the invention can also include hybrid and modified forms of TrnR2 and fragments thereof in which certain amino acids have been deleted or replaced and modifications such as where one or more amino acids have been changed to a modified amino acid or unusual amino acid and modifications such as glycosylations so long as the hybrid or modified form retains TrnR2 biological activity. By TrnR2 biological activity, it is meant that Ret PTK is activated in the presence of the hybrid or modified TrnR2 and NTN or GDNF or other TRN growth factor, although not necessarily at the same level of potency as that of TrnR2 isolated from tissues or cells which naturally produce TrnR2 such as SCG neurons or that of the recombinantly produced human or mouse TrnR2.

Also included within the meaning of substantially homologous is any TrnR2 polypeptide which may be isolated by virtue of cross-reactivity with antibodies to the TrnR2 described herein or whose encoding nucleotide sequences including genomic DNA, mRNA or cDNA may be isolated through degenerate PCR or by hybridization with the complementary sequence of genomic or subgenomic WO 98/46622 PCT/US98/07996 nucleotide sequences or cDNA of the human or mouse TrnR2 described herein or fragments thereof. It will also be appreciated by one skilled in the art that allelic variants of TrnR2 are included within the present invention.

Isolation of cDNAs corresponding to two alternatively spliced TrnR2 mRNAs of different lengths indicate a TrnR2 protein product for each spliced variant may exist. Thus, any and all TrnR2 proteins encoded by alternatively spliced TrnR2 mRNAs are intended to be included within the term "TrnR2 polypeptide" as used herein.

The predicted amino acid sequence and biological function of TrnR2 indicate that it is an externally disposed plasma membrane protein anchored to the extracellular surface of the cell membrane by a glycosylphosphatidyl (GPI) linkage. It is well-known in the art that GPI anchored membrane proteins are synthesized as a precursor protein with an N-terminal signal sequence and a C-terminal GPIsp which are cleaved during the cellular processing events leading to the mature protein. Thus, all precursor TrnR2 proteins containing either or both of the signal sequence and GPIsp as well as mature TrnR2 proteins which contain neither signal peptide are embraced by the term "TrnR2 polypeptide". It is also well-recognized in the art that GPI anchored proteins may exist in soluble form following cleavage of the GPI linkage with phospholipases. Therefore, the term "TrnR2 polypeptide also includes soluble TrnR2 polypeptides generated by phospholipase cleavage of anchored TrnR2 polypeptides.

A preferred TrnR2 polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:6. A more preferred TrnR2 polypeptide is human mature TrnR2 protein which has the amino acid sequence set forth in SEQ ID WO 98/46622 PCT/US98/07996 21 NO:3.

The term "TrnR2 polypeptide" is intended to include fragments which have one or more of the biological activities of precursor or mature TrnR2 protein. Such activities include binding a member of the TRN growth factor family, particularly NTN or GDNF, and binding to Ret in the presence of NTN, GDNF, or other TRN family member, with such binding leading to Ret phosphorylation.

It is believed that by using the nucleotide sequences encoding precursor TrnR2, which are provided herein, those skilled in the art can readily construct multiple TrnR2 fragments and screen them for the desired biological activity.

A preferred TrnR2 fragment is one which lacks the hydrophobic domain for the GPI-attachment site which are referred to herein as soluble TrnR2 fragments. For example, soluble TrnR2 fragments include, but are not limited to, polypeptides having an amino acid sequence encoded by nucleotide residues 99 to 1331 of SEQ ID NO:1 (human long splice variant), nucleotides 36-74 and 474- 1331 of SEQ ID NO:1 (human short splice variant), nucleotide residues 64-1296 of SEQ ID NO:4 (mouse long splice varaint), nucleotides 1-39 and 438-1296 (mouse short splice variant), and fragments there.

TrnR2 fragments also included in the scope of the invention are antigenic fragments which are capable of eliciting TrnR2 specific antibodies when administered to a host animal as conjugated to a carrier molecule or in nonconjugated form.

A TrnR2 protein of the present invention may be isolated in purified form from tissues or cells which naturally produce TrnR2. Such tissues or cells may originate from any eukaryotic organism that naturally produce TrnR2. Alternatively, a substantially pure TrnR2 polypeptide may be prepared by recombinant

DNA

technology. By "pure form" or "purified form" or WO 98/46622 PCT/US98/07996 22 "substantially purified form" it is meant that a TrnR2 composition is substantially free of other proteins which are not TrnR2.

One skilled in the art can readily follow known methods for isolating proteins in order to obtain the TrnR2 polypeptide substantially free of other proteins, including immunochromatography, size-exclusion chromatography, HPLC, ion-exchange chromatography, and ligand affinity chromatography. As readily appreciated by those skilled in the art, an example of one way to obtain TrnR2 protein naturally produced by cells in culture would be to treat the cells with PI-PLC to cleave the GPI-linked TrnR2 protein from the cell surface, and then purifying the soluble TrnR2 protein from the media by ligand affinity chromatography using NTN or GDNF as the ligand or by immunochromatography using an antibody raised against TrnR2 protein or an antigenic TrnR2 peptide.

A recombinant TrnR2 polypeptide may be made by expressing the DNA sequences encoding TrnR2 in a suitable transformed host cell. Using methods well known in the art, the DNA encoding the TrnR2 polypeptide may be linked to an expression vector, transformed into a host cell and conditions established that are suitable for expression of the TrnR2 polypeptide by the transformed cell.

Any suitable expression vector may be employed to produce recombinant TrnR2 such as, for example, the mammalian expression vector pCMV-neo (Brewer, Meth.Cell Biol. 43:233-245, 1994, incorporated herein by reference) which was used herein or the E. coli pET expression vectors, in particular, pET-30a (Studier et al., Methods Enzymol. 185:60-89, 1990 which is incorporated by reference). Other suitable expression vectors for expression in mammalian and bacterial cells are known in the art as are expression vectors for use in yeast or insect cells. Baculovirus expression systems can also be WO 98/46622 PCT/US98/07996 23 employed.

In another embodiment, the present invention provides an isolated and purified polynucleotide comprising a nucleotide sequence that encodes a TrnR2 polypeptide. Nucleotide sequences included in the invention are those encoding human or mouse precursor and mature TrnR2 proteins. Preferred nucleotide sequences encoding human proteins are as set forth in SEQ ID NO:1: nucleotides 36-1427 encode a precursor TrnR2 and nucleotides 99-1331 encode a human mature TrnR2.

Similarly, preferred nucleotide sequences which encode a mouse precursor and a mouse mature protein are nucleotides 1-1389 and nucleotides 64-1296 of SEQ ID NO:4, respectively. Nucleotide sequences encoding TrnR2 fragments are also contemplated, particularly soluble TrnR2 fragments. Preferred nucleotide sequences encoding a soluble TrnR2 fragment are residues 99 to 1331 of SEQ ID NO:1 (human long splice variant), nucleotides 36-74 and 474-1427 of SEQ ID NO:1 (human short splice variant), residues 64-1296 of SEQ ID NO:4 (mouse long splice variant) and nucleotides 1-39 and 438-1389 of SEQ ID NO:4 (mouse short splice variant). It is understood by the skilled artisan that degenerate DNA sequences can encode the TrnR2 polypeptides described herein and these are also intended to be included within the present invention.

Based upon the high sequence conservation between the human and mouse TrnR2 coding sequences, it is believed that DNA probes and primers can be made and used to readily obtain TrnR2-encoding cDNA clones from different species. Thus, a cDNA encoding a TrnR2 from a species other than human or mouse is embraced by the invention.

Also included within the scope of this invention are nucleotide sequences that are substantially the same as a nucleic acid sequence encoding TrnR2. Substantially the WO 98/46622 PCT/US98/07996 24 same sequences may, for example, be substituted with codons more readily expressed in a given host cell such as E. coli according to well known and standard procedures. Such modified nucleic acid sequences would be included within the scope of this invention.

Specific nucleic acid sequences can be modified by those skilled in the art and, thus, all nucleic acid sequences which encode for the amino acid sequences of TrnR2 or biologically active fragments thereof can likewise be so modified. The present invention thus also includes polynucleotides containing a nucleic acid sequence which will hybridize with all such nucleic acid sequences or complements of the nucleic acid sequences where appropriate and encode for a polypeptide having biological activity as a coreceptor for NTN or GDNF. The present invention also includes nucleic acid sequences which encode for polypeptides that have one or more of the biological activities of TrnR2 and those that are recognized by antibodies that bind to TrnR2.

The cDNA sequences provided herein allow genomic clones for the TrnR2 gene to be readily isolated. One use for genomic clones is for chromosome localization studies. For example, human and mouse genomic clones for the TrnR2 gene were obtained by screening P1 (mouse) and PAC (human) genomic libraries (Genome Systems, St. Louis, MO) with a PCR assay using primers derived from the TrnR2 coding region. A human PAC genomic clone containing the TrnR2 gene in a 120 kb genomic fragment was used to localize the TrnR2 gene to the short arm of human chromosome 8 in region p12-2 1 by fluorescence in situ hybridization analysis (FISH). The mouse chromosomal location can readily be determined in a similar fashion using a mouse genomic clone.

A search of the database for neurological diseases genetically mapped to the human locus revealed only one such disease, SPG5A, an autosomal recessive form of WO 98/46622 PCT/US98/07996 spastic paraplegia, localized to the paracentric region of chromosome 8 (Hentati et al., Hum.Molec.Genet. 3:1263- 1267, 1994, incorporated herein by reference). Also, an amplification event on 8pl2 has been observed in some cases of breast and ovarian cancer (Imbert et al., Genomics 32:29-38, 1996, incorporated herein by reference). Genomic clones for the TrnR2 gene are also useful for surveying for possible gene or chromosome rearrangements in patients suffering from a neurological disease with no identified cause.

The present invention also encompasses vectors comprising expression regulatory elements operably linked to any of the nucleic acid sequences included within the scope of the invention. This invention also includes host cells, of any variety, that have been transformed with vectors comprising expression regulatory elements operably linked to any of the nucleic acid sequences included within the scope of the present invention.

In one embodiment, recombinant cells expressing both Ret and TrnR2 are provided which are useful for screening compounds for TRN growth factor agonistic or antagonistic activity. The recombinant cells may be produced by transforming a suitable host cell such as fibroblasts with nucleotide sequences encoding for expression Ret and TrnR2 proteins. The protein-encoding nucleotide sequences may be on the same or on different vectors.

Ret-encoding nucleotide sequences may be readily isolated by screening a suitable cDNA library using an oligonucleotide probe corresponding to a region of the known human and/or mouse amino acid sequences (Iwamoto, et al., Oncogene 8, 1087-1091, 1993 incorporated herein by reference). Suitable cDNA libraries would be those prepared from tissues known to express Ret, including but not limited to placental tissue.

Agonistic or antagonistic activity of a test compound would be determined by incubating the target WO 98/46622 PCT/US98/07996 26 Ret/TrnR2-expressing cells with the test compound in the absence or presence of a TRN growth factor such as NTN, GDNF, or persephin and assaying for Ret protein tyrosine kinase activity. Compounds which increase Ret PTK activity in the absence of a TRN have agonistic activity, while compounds which reduce or block Ret PTK activity in the presence of a TRN are TRN antagonists.

The Ret PTK activity may be assayed by looking for tyrosine phosphorylation of Ret as described herein.

Alternatively, or additionally, the target cell may be engineered to include a reporter gene whose expression is under the control of a TRN-responsive enhancer/promoter region. NTN and GDNF are known to cause an increase in mitogen-activated protein kinase (MAPK) activation in SCG neurons (Kotzbauer, et al., Nature 384:467-470, 1996 incorporated herein by reference) and the inventors herein have also discovered that phosphatidylinositol 3kinase (PI-3-K) is also activated by NTN and GDNF. Thus, expression of a reporter gene operably linked to the enhancer/promoter regions of genes downstream in the MAPK or PI-3-K intracellular signalling pathways would be expected to be increased in the presence of a TRN agonist and decreased in the presence of a TRN antagonist. It is believed that such enhancer/promoter regions are known to those skilled in the art and can be readily isolated.

Known reporter genes which encode for readily detectable products include, but are not limited to, Pgalactosidase, chloramphenicol acetyl transferase, luciferase and p-glucuronidase. Detection of the expression of known reporter genes, which is well known to those skilled in the art, may serve as a sensitive indicator for any NTN or GDNF agonistic activity of test compounds.

Methods are also provided herein for producing TrnR2 polypeptides. Preparation can be by isolation from a variety of cell types so long as the cell type expresses WO 98/46622 PCT/US98/07996 27 TrnR2 protein. Examples of biological material suitable for TrnR2 isolation include, but are not limited to, brain tissue, human neuroblastoma cell lines, and superior cervical ganglion cells. A second and preferred method involves utilization of recombinant methods by isolating a nucleic acid sequence encoding a TrnR2 polypeptide, cloning the sequence along with appropriate regulatory sequences into suitable vectors and cell types, and expressing the sequence to produce TrnR2. In one embodiment, the nucleotide sequence does not encode the C-terminal hydrophobic domain containing the GPIattachment site, thus producing a soluble TrnR2 fragment that is secreted into the growth medium.

The present invention also provides probes which may be used to identify cells and tissues which may be responsive to NTN or GDNF in normal or disease conditions by detecting TrnR2 expression in such cells. Detection of TrnR2 expression may also be useful to determine if a patient suffering from a NTN- or GDNF-related disorder has aberrant TrnR2 expression or expresses a biologically inactive TrnR2 mutant. TrnR2 expression may be detected with probes which react with TrnR2 mRNA or TrnR2 protein.

For example, to detect the presence of mRNA encoding a TrnR2 polypeptide or biologically inactive mutant thereof, a sample is obtained from a patient. The sample may be from blood or a tissue biopsy. The sample may be treated to extract the nucleic acids contained therein which may then be subjected to gel electrophoresis or other size separation techniques.

The mRNA of the sample is contacted with a polynucleotide probe comprising a nucleic acid sequence complementary to TrnR2 mRNA. The polynucleotide probe may be an oligonucleotide containing a minimum of about 8 to 12, preferably at least about 20, contiguous nucleotides which are complementary to the TrnR2 target sequence. Oligonucleotide probes may be prepared by any WO 98/46622 PCT/US98/07996 28 method known in the art such as, for example, excision, transcription or chemical synthesis. Alternatively, the polynucleotide probe may comprise a cDNA encoding TrnR2 or a fragment thereof as a probe.

To enable detection of hybridization between the polynucleotide probe and the target sequence, the probe may be labelled with any detectable label known in the art such as, for example, radioactive or fluorescent labels or enzymatic markers. Labeling of the probe can be accomplished by any method known in the art such as by PCR, random priming, end labelling, nick translation or the like. One skilled in the art will also recognize that other methods not employing a labelled probe can be used to determine the hybridization. Examples of methods that can be used for detecting hybridization include Southern blotting, fluorescence in situ hybridization, and single-strand conformation polymorphism with PCR amplification.

Hybridization conditions for the type of probe used may be readily determined by those skilled in the art.

High stringency conditions are preferred in order to prevent false positives. The stringency of hybridization is determined by a number of factors in the hybridization and washing steps. Such factors are well known to those skilled in the art and outlined in, for example, Sambrook et al. (Sambrook, et al., 1989, supra).

The sensitivity of detection in a sample of TrnR2 mRNA may be increased using the technique of reverse transcription/polymerization chain reaction (RT/PCR) to amplify cDNA transcribed from TrnR2 mRNA using primers specific for a TrnR2-encoding nucleotide sequence (see example 4 and Fig. 6 below). The method of RT/PCR is well known and routinely performed by those skilled in the art.

The present invention further provides for methods to detect the presence of the TrnR2 protein or WO 98/46622 PCT/US98/07996 29 biologically inactive mutants thereof in a sample obtained from a patient. Any method known in the art for detecting proteins can be used. Such methods include, but are not limited to immunodiffusion, immunoelectrophoresis, immunochemical methods, binderligand assays, immunohistochemical techniques, agglutination and complement assays. (For example, see Basic and Clinical Immunology, Sites and Terr, eds., Appleton Lange, Norwalk, Conn. pp 217-262, 1991 which is incorporated by reference). Preferred are binderligand immunoassay methods which involve reacting antibodies with an epitope or epitopes of a TrnR2 protein or derivative thereof to competitively displace a labeled TrnR2 polypeptide.

As used herein, a derivative of the TrnR2 protein is intended to include a polypeptide in which certain amino acids have been deleted or replaced or changed to modified or unusual amino acids wherein the derivative is biologically equivalent to TrnR2 and wherein the polypeptide derivative cross-reacts with antibodies raised against the TrnR2 protein. By cross-reaction it is meant that an antibody reacts with an antigen other than the one that induced its formation.

Numerous competitive and non-competitive protein binding immunoassays are well known in the art.

Antibodies as used herein are intended to include fulllength anti-TrnR2 antibody molecules and TrnR2 binding fragments of such antibody molecules. The anti-TrnR2 antibody may be unlabeled, for example as used in agglutination tests, or labeled for use in a wide variety of assay methods. Labels that can be used include radionuclides, enzymes, fluorescers, chemiluminescers, enzyme substrates or co-factors, enzyme inhibitors, particles, dyes and the like for use in radioimmunoassay (RIA), enzyme immunoassays, enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassays and WO 98/46622 PCT/US98/07996 the like.

Polyclonal or monoclonal antibodies to the TrnR2 protein or an epitope thereof can be made for use in immunoassays by any of a number of methods known in the art. By epitope reference is made to an antigenic determinant of a polypeptide. An epitope could comprise 3 amino acids in a spacial conformation which is unique to the epitope. Generally an epitope consists of at least 5 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, x-ray crystallography and 2 dimensional nuclear magnetic resonance.

One approach for preparing antibodies to a protein is the selection and preparation of an amino acid sequence of all or part of the protein, chemically synthesizing the sequence and injecting it into an appropriate animal, usually a rabbit or a mouse (See Example 11).

Oligopeptides can be selected as candidates for the production of an antibody to the TrnR2 protein based upon the oligopeptides lying in hydrophilic regions, which are thus likely to be exposed in the mature protein.

Antibodies to TrnR2 can also be raised against oligopeptides that include one or more of the conserved regions identified herein such that the antibody can cross-react with other family members. Such antibodies can be used to identify and isolate the other family members.

Methods for preparation of the TrnR2 protein or an epitope thereof include, but are not limited to chemical synthesis, recombinant DNA techniques or isolation from biological samples. Chemical synthesis of a peptide can be performed, for example, by the classical Merrifeld method of solid phase peptide synthesis (Merrifeld, J Am Chem Soc 85:2149, 1963 which is incorporated by reference) or the FMOC strategy on a Rapid Automated WO 98/46622 PCT/US98/07996 31 Multiple Peptide Synthesis system (DuPont Company, Wilmington, DE) (Caprino and Han, J Org Chem 37:3404, 1972 which is incorporated by reference).

Polyclonal antibodies can be prepared by immunizing rabbits or other animals by injecting antigen followed by subsequent boosts at appropriate intervals. The animals are bled and sera assayed against purified TrnR2 protein, usually by ELISA or by bioassay based upon the ability to block one or more of the biological activities of TrnR2.

When using avian species, e.g. chicken, turkey and the like, the antibody can be isolated from the yolk of the egg. Monoclonal antibodies can be prepared after the method of Milstein and Kohler by fusing splenocytes from immunized mice with continuously replicating tumor cells such as myeloma or lymphoma cells. (Milstein and Kohler Nature 256:495-497, 1975; Gulfre and Milstein, Methods in Enzymology: Immunochemical Techniques 73:1-46, Langone and Banatis eds., Academic Press, 1981 which are incorporated by reference). The hybridoma cells so formed are then cloned by limiting dilution methods and supernates assayed for antibody production by ELISA, RIA or bioassay.

If a patient suffering from a GDNF or NTN-related disorder expresses apparently normal levels of TrnR2, it may be possible that the expressed TrnR2 may be a biologically inactive mutant. To verify this, cDNA obtained from mRNA isolated from a sample of a relevant target tissue may be sequenced using methods known in the art.

The present invention also includes therapeutic or pharmaceutical compositions comprising an effective amount of a TrnR2 polypeptide for treating patients with cellular degeneration and a method for promoting cell survival which comprises administering to a patient in need thereof a therapeutically effective amount of a TrnR2 polypeptide.

WO 98/46622 PCT/US98/07996 32 Certain degeneration disorders may be related to a lack of or reduced expression of biologically active TrnR2, while NTN or GDNF expression is normal. In addition, it may be desirable under certain circumstances to increase TrnR2 levels even where TrnR2 expression is not decreased. It has been shown that soluble TrnRl added to Ret-expressing cells, the TrnRl is not bound to the membrane, can activate Ret in the presence of GDNF (Jing et al, supra). Thus, it is believed that administering a TrnR2 polypeptide will increase the number of cells which have a functional TrnR2/Ret receptor complex and thus capable of responding to endogenously produced NTN and/or GDNF.

Additional survival or growth promoting effects may be achieved by administering NTN and/or GDNF along with the TrnR2 polypeptide. It is believed that treatment with one or both of these growth factors together with a TrnR co-receptor would increase the sensitivity of cells normally responsive to the growth factor(s). In addition, such treatment would be expected to promote the survival or growth of other cell types that express Ret but that are not normally responsive to NTN or GDNF.

Alternatively, expression of TrnR2 could be increased in tissues defective in such expression by gene therapy. Patients may be implanted with vectors or cells capable of producing a biologically-active TrnR2 polypeptide. In one approach, cells that secrete soluble TrnR2 may be encapsulated into semipermeable membranes for implantation into a patient. The cells can be those that normally express a TrnR2 protein or the cells can be transformed to express a TrnR2 polypeptide. When the patient is human, it is preferred that the TrnR2 be human TrnR2. However, the formulations and methods herein can be used for veterinary as well as human applications and the term "patient" as used herein is intended to include human and veterinary patients.

WO 98/46622 PCT/US98/07996 33 Cells can be grown ex vivo, for example, for use in transplantation or engraftment into patients (Muench et al., Leuk Lymph 16:1-11, 1994 which is incorporated by reference). TrnR2 in combination with NTN or GDNF can be administered to such cells to elicit growth and differentiation, provided the cells express Ret. Ret expression has been observed during embryogenesis in many cell lineages of the developing central and peripheral nervous systems. Ret has also been detected outside the nervous system as well, including gut and kidney. Thus, in another embodiment of the present invention, a composition comprising TrnR2 and NTN or GDNF is used to promote the ex vivo expansion of Ret-expressing cells for transplantation or engraftment.

These compositions and methods are useful for treating a number of degenerative diseases. Where the cellular degeneration involves neuronal degeneration, the diseases include, but are not limited to peripheral neuropathy, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, ischemic stroke, acute brain injury, acute spinal chord injury, nervous system tumors, multiple sclerosis, peripheral nerve trauma or injury, exposure to neurotoxins, metabolic diseases such as diabetes or renal dysfunctions and damage caused by infectious agents.

Where the cellular degeneration involves bone marrow cell degeneration, the diseases include, but are not limited to disorders of insufficient blood cells such as, for example, leukopenias including eosinopenia and/or basopenia, lymphopenia, monocytopenia, neutropenia, anemias, thrombocytopenia as well as an insufficiency of stem cells for any of the above. The above cells and tissues can also be treated for depressed function.

The compositions and methods herein can also be useful to prevent degeneration and/or promote survival in other non-neuronal tissues as well. One skilled in the WO 98/46622 PCT/US98/07996 34 art can readily determine using a variety of assays known in the art whether a particular cell type expresses Ret and would thus likely be activated in the presence of TrnR2 and a TRN such as NTN or GDNF.

In certain circumstances, it may be desirable to modulate or decrease the trophic effect of endogenously synthesized TRN growth factors, including NTN and/or GDNF. This may be achieved by blocking binding of the growth factor to its receptor in the target tissue or by decreasing TrnR2 expression in the target tissue. Thus, appropriate treatments, for example, may involve administration of TrnR2 antibodies or other compounds having TRN antagonist properties, or the use of antisense polynucleotides to modulate TrnR2 expression.

Specific antibodies, either polyclonal or monoclonal, may be capable of preventing binding of NTN an/or GDNF to TrnR2 or, alternatively, may prevent the formation of a functional TrnR2/Ret receptor complex.

Such antibodies can be produced by any suitable method known in the art. For example, murine or human monoclonal antibodies can be produced by hybridoma technology or by combinatorial antibody library technology, including panning a phage display library.

The antibody may be engineered using recombinant techniques to produce an antibody with desirable characteristics such as being "humanized" to be better tolerated by the patient or having specificities for both TrnR2 and Ret or both TrnR2 and a TRN growth factor.

Such antibody engineering techniques are known in the art. See for example, Hayden et al., Curr. Opin.

Immunol. 9(2):201-212, 1997, incorporated herein by reference. Alternatively, the TrnR2 protein, or an immunologically active fragment thereof, or an antiidiotypic antibody, or fragment thereof can be administered to an animal to elicit the production of antibodies capable of recognizing and binding to the WO 98/46622 PCT/US98/07996 TrnR2 protein. Such antibodies can be from any class of antibodies including, but not limited to IgG, IgA, IgM, IgD, and IgE or in the case of avian species, IgY and from any subclass of antibodies.

It is also envisioned that soluble TrnR2 polypeptides and fragments can also serve as TRN antagonists. For example, it is believed that a soluble TrnR2 administered in excess would GDNF, NTN, and possibly other TRN growth factors, thereby sequestering the TRN growth factor from the anchored TrnR2 receptors on target cells. Similarly, if excessive levels of a TRN growth factor, particularly GDNF or NTN, was circulating, administration of soluble TrnR2 may act to sequester the growth factor in the plasma and possibly facilitate their excretion, thereby limiting the effects of the growth factor in the body.

In another aspect of the present invention, TrnR2 antisense oligonucleotides can be made and a method utilized for diminishing the level of expression of TrnR2 protein by a cell comprising administering one or more TrnR2 antisense oligonucleotides. By TrnR2 antisense oligonucleotides reference is made to oligonucleotides that have a nucleotide sequence that interacts through base pairing with a specific complementary nucleic acid sequence involved in the expression of TrnR2 such that the expression of TrnR2 is reduced. Preferably, the specific nucleic acid sequence involved in the expression of TrnR2 is contained within a genomic DNA molecule or mRNA molecule that encodes TnrR2. A genomic DNA molecule may comprise regulatory regions of the TrnR2 gene and/or coding sequences for precursor or mature TrnR2 protein.

The term complementary to a nucleotide sequence in the context of TrnR2 antisense oligonucleotides and methods therefor means sufficiently complementary to such a sequence as to allow hybridization to that sequence in a cell, under physiological conditions. The TrnR2 WO 98/46622 PCT/US98/07996 36 antisense oligonucleotides preferably comprise a sequence containing from about 8 to about 100 nucleotides and more preferably the TrnR2 antisense oligonucleotides comprise from about 15 to about 30 nucleotides.

The TrnR2 antisense oligonucleotides can also include derivatives which contain a variety of modifications that confer resistance to nucleolytic degradation such as, for example, modified internucleoside linkages modified nucleic acid bases and/or sugars and the like (Uhlmann and Peyman, Chemical Reviews 90:543-584, 1990; Schneider and Banner, Tetrahedron Lett 31:335, 1990; Milligan et al., J Med Chem 36:1923-1937, 1993; Tseng et al., Cancer Gene Therap 1:65-71, 1994; Miller et al., Parasitology 10:92-97, 1994 which are incorporated by reference). Such derivatives include but are not limited to backbone modifications such as phosphotriester, phosphorothioate, methylphosphonate, phosphoramidate, phosphorodithioate and formacetal as well as morpholino, peptide nucleic acid analogue and dithioate repeating units.

The TrnR2 antisense polynucleotides of the present invention can be used in treating overexpression of TrnR2 or reduce sensitivity of cells to inappropriate expression of NTN or GDNF. Such treatment can also include the ex vivo treatment of cells.

The therapeutic or pharmaceutical compositions of the present invention can be administered by any suitable route known in the art including for example intravenous, subcutaneous, intramuscular, transdermal, intrathecal or intracerebral or administration to cells in ex vivo treatment protocols. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulation.

For treating tissues in the central nervous system, administration can be by injection or infusion into the cerebrospinal fluid (CSF). When it is intended that WO 98/46622 PCT/US98/07996 37 TrnR2 be administered to cells in the central nervous system, administration can be by intravenous injection with one or more agents capable of promoting penetration of TrnR2 across the blood-brain barrier such as an antibody to the transferrin receptor. Co-administration may comprise physically coupling any known blood-brain penetrating agent to TrnR2. (See for example, Friden et al., Science 259:373-377, 1993 which is incorporated by reference).

A TrnR2 polypeptide can also be linked or conjugated with agents that provide other desirable pharmaceutical or pharmacodynamic properties. For example, a TrnR2 polypeptide can be stably linked to a polymer such as polyethylene glycol to obtain desirable properties of solubility, stability, half-life and other pharmaceutically advantageous properties. (See for example Davis et al. Enzyme Eng 4:169-73, 1978; Burnham, Am J Hosp Pharm 51:210-218, 1994 which are incorporated by reference).

The compositions are usually employed in the form of pharmaceutical preparations. Such preparations are made in a manner well known in the pharmaceutical art. One preferred preparation utilizes a vehicle of physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, sterile water or the like may also be used. It may also be desirable that a suitable buffer be present in the composition. Such solutions can, if desired, be lyophilized and stored in a sterile ampoule ready for reconstitution by the addition of sterile water for ready injection. The primary solvent can be aqueous or alternatively non-aqueous.

TrnR2 can also be incorporated into a solid or semi-solid biologically compatible matrix which can be implanted into tissues requiring treatment.

WO 98/46622 PCT/US98/07996 38 The carrier can also contain other pharmaceuticallyacceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation. Similarly, the carrier may contain still other pharmaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-brain barrier. Such excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion into the cerebrospinal fluid by continuous or periodic infusion.

Dose administration can be repeated depending upon the pharmacokinetic parameters of the dosage formulation and the route of administration used.

It is also contemplated that certain formulations containing TrnR2 are to be administered orally. Such formulations are preferably encapsulated and formulated with suitable carriers in solid dosage forms. Some examples of suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like.

The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents. The compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art. The formulations can also contain substances that diminish proteolytic degradation and promote absorption such as, WO 98/46622 PCT/US98/07996 39 for example, surface active agents.

The specific dose is calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied. The dose will also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. It is believed that such calculations can be readily made by one skilled in the art in light of the dose-response curves disclosed herein for NTN- or GDNF-induced Ret activation in TrnR2/Ret-expressing cells. Exact dosages are determined in conjunction with standard dose-response studies. It will be understood that the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration.

The invention also provides the identification of a novel receptor gene family for TRN neurotrophic factors.

The known members of this family, Trnl and TrnR2, share approximately 48% percent amino acid sequence identity and about 53% sequence homology. The inventors herein believe that other unidentified genes may exist that encode proteins that have substantial amino acid sequence homology to TrnRl and TrnR2 and which function as receptors for growth factors selective for the same or different tissues having the same or different biological activities. A different spectrum of activity with respect to tissues affected and/or response elicited could result from preferential activation of different receptors by different family members as is known to WO 98/46622 PCT/US98/07996 occur with members of the NGF family of neurotrophic factors and Trk receptors (Tuszynski and Gage, supra).

As a consequence of members of a particular gene family showing substantial conservation of amino acid sequence among the protein products of the family members, there is considerable conservation of sequences at the DNA level. This forms the basis for a new approach for identifying other members of the receptor gene family to which TrnRl and TrnR2 belong. The method used for such identification is cross-hybridization using nucleic acid probes derived from one family member to form stable hybrid duplex molecules with homologous sequences from different members of the gene family or to amplify nucleic acid sequences from different family members. (See for example, Kaisho et al. FEBS Letters 266:187-191, 1990 which is incorporated by reference).

The sequence from the different family member may not be identical to the probe, but will, nevertheless be sufficiently related to the probe sequence to hybridize with the probe. Alternatively, PCR using primers from one family member can be used to amplify homologous sequences in additional family members.

The above approaches would not have heretofore been successful in identifying other gene family members because only one family member, TrnRl (GDNFR-a) was known. With the identification of TrnR2 herein, however, unique new probes and primers can be made that contain sequences from the longer conserved regions of this gene family (see boxed regions in Figure The new probes and primers made available from the present work make possible this powerful new approach which can now successfully identify other gene family members. Using this new approach, one may screen for genes related to TrnRl and TrnR2 in amino acid sequence homology by preparing DNA or RNA probes based upon the conserved regions in the TrnRl and TrnR2 proteins.

WO 98/46622 PCT/US98/07996 41 Therefore, one embodiment of the present invention comprises probes and primers that are unique to or derived from a nucleotide sequence encoding such conserved regions and a method for identifying further members of the TrnR gene family. Examples of such conserved region amino acid sequences include but are not limited to Cys-Arg-Cys-Lys-Arg-Gly-Met-Lys-Lys-Glu

(SEQ

ID NO:9); Cys-Asn-Arg-Arg-Lys-Cys-His-Lys-Ala-Lys-Arg (SEQ ID NO:10), and Cys-Leu-Xaa-Asn-Ala-Ile-Glu-Ala-Phe- Gly-Asn-Gly (SEQ ID NO:11) where Xaa is Lys or Arg.

Degenerate oligonucleotides containing all of the possible nucleotide sequences which code for one or more of the TrnR2 conserved amino acid sequences can be synthesized for use as hybridization probes or amplification primers. The nucleotide sequence may be based on the above listed conserved sequences or chosen from the other boxed conserved regions shown in Figure 1.

To reduce the number of different oligonucleotides in a degenerate mix, an inosine base, or another "universal" base, can be incorporated in the synthesis at positions where all four nucleotides are possible. Univeral bases such as inosine form base pairs with each of the four normal DNA bases which are less stabilizing than AT and GC base pairs but which are also less destabilizing than mismatches between the normal bases AG, AC, TG,

TC).

Sources of nucleic acid for screening would include mammalian genomic DNA, cDNA reversed transcribed from mRNA obtained from mammalian cells, or genomic or cDNA libraries prepared from mammalian species cloned into any suitable vector.

Hybridization using the new probes to conserved regions of the nucleic acid sequences would be performed under reduced stringency conditions. Factors involved in determining stringency conditions are well known in the art (for example, see Sambrook et al., Molecular Cloning, WO 98/46622 PCT/US98/07996 42 2nd Ed., 1989 which is incorporated by reference).

Sources of nucleic acid for screening would include genomic DNA libraries from mammalian species or cDNA libraries constructed using RNA obtained from mammalian cells cloned into any suitable vector.

PCR primers would be utilized under PCR conditions of reduced annealing temperature which would allow amplification of sequences from gene family members other than TrnRl and TrnR2. To identify the sequences of these products, they can be gel purified and ligated into any suitable cloning vector and transformed into bacteria.

The resulting clones can be screened with an oligonucleotide probe for either a unique TrnRl or a unique TrnR2 sequence in the amplified region. Clones not hybridizing to either unique probe can be sequenced and if found to encode previously unisolated family members, the sequence of that clone can be used to isolate full length cDNA clones and genomic clones.

A

similar method was used to isolate new gene members (GDF-3 and GDF-9) of the TGF-B superfamily based on homology between previously identified genes (McPherron

J

Biol Chem 268: 3444-3449, 1993 which is incorporated by reference).

Alternatively, other TrnR family members may be identified and/or obtained by screening a cDNA expression library for the presence of proteins cross-reacting with an antibody capable of reacting with a polypeptide containing a TrnR conserved region, an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11. Preparation of cDNA libraries in mammalian expression systems is known in the art (see Jing et al., supra, Treanor et al., supra, Gearing et al., EMBO J. 8:3667-3676, 1989, and Takebe, et al., Mol. Cell. Biol. 8:466-472, 1988, incorporated herein by reference). A clone expressing a polypeptide that binds to an anti-TrnR conserved region would be WO 98/46622 PCT/US98/07996 43 isolated and its cDNAs sequenced to determine whether it encoded a TrnR family member based on comparing its predicted amino acid sequences with those of TrnRl and TrnR2.

Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein.

It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples.

Example 1 This example illustrates that either TrnRl or TrnR2 can mediate the signaling of NTN or GDNF through the Ret protein tyrosine kinase.

Generation of fibroblasts expressing Ret and TrnRl or TrnR2 To examine the possibility that TrnRl or TrnR2 can form a functional receptor complex with Ret for NTN and/or GDNF, NIH3T3 fibroblasts which stably express Ret alone, both Ret and TrnRl, or both Ret and TrnR2 were generated.

Briefly, full length human Ret cDNA (gift of Dr. H.

Donnis-Keller, Washington University, St. Louis, MO) was subcloned into the pCMV-Neo vector (Brewer, Meth.

Cell Biol. 43:233-245, 1994, incorporated herein by reference). NIH3T3 cells (subclone MG87; Zhan et al., 1987) were transfected with the Ret-CMV-neo plasmid, grown in DMEM plus 10% fetal bovine serum (Hyclone), and stable transfectants expressing Ret were selected with 1 mg/ml G418. Positive clones were screened for Ret expression on immunoblots probed with an anti-Ret antibody (Santa Cruz Biotechnology, Inc., Santa Cruz,

CA).

WO 98/46622 PCT/US98/07996 44 A clonal Ret-expressing cell line was used as the parent to generate TrnRl/Ret and TrnR2/Ret expressing cells by transfection with TrnRl or TrnR2 expression constructs. To prepare the TrnRl expression construct, a TrnRl cDNA was obtained from a rat postnatal-day-i library by Klentaq LA PCR with the primers: GCGGTACCATGTTCCTAGCCACTCTGTACTTCGC-3' (SEQ ID NO:14) and 5'-GCTCTAGACTACGACGTTTCTGCCAACGATACAG-3' (SEQ ID The amplified product was cloned into the EcoRV site of pBluescript KS (Stratagene, La Jolla, CA, sequenced and subcloned into the HindIII and BamHI sites of pCMV-Neo (Brewer, 1994). For the TrnR2 expression construct, the coding region of the long form of human TrnR2 cDNA was amplified from the same Marathon RACE human brain cDNA library used to clone and sequence TrnR2. Amplification primers were 5'-GCGGTACCATGATCTTGGCAAACGTCTGC- 3 (SEQ ID NO:16) and 5'-GCTCTAGAGTCAGGCGGCTGTTCTTGTCTGCG-3' (SEQ ID NO:17). The product was cloned into pCMV-neo and the insert confirmed by sequencing.

The TrnRl-CMV-Neo plasmid or the TrnR2 CMV-Neo plasmid was co-transfected with SV2-HisD (gift of Dr.

Richard Mulligan, Massachusetts Institute of Technology) into the Ret-expressing 3T3 cells and double transfectants selected in 2 mM L-histidinol (Sigma, St.

Louis, MO). TrnRl- and TrnR2-expressing clones were confirmed by western (Ret) and northern (TrnRl or TrnR2) blotting.

Preparation of recombinant NTN and GDNF A synthetic gene for the mature mouse NTN coding sequence was prepared from four partially overlapping oligonucleotides containing the Eschericia coli coli) codon preferences: 5'-GCA TAT GCC GGG TGC TCG TCC GTG CGG CCT GCG TGC AAC TGG AAG TTC GTG TTT CTG AAC TGG GTC TGG GTT ACA CTT CTG ACG AAA CTG T-3'(SEQ ID NO:18); GAC GCA GAC GAC GCA GAC CCA GGT CGT AGA TAC GGA TAG CAG CTT CGC ATG CAC CAG CGC AGT AAC GGA ACA GAA CAG TTT CGT- WO 98/46622 PCT/US98/07996 3' (SEQ ID NO:1 9 5'-CTG CGT CAG CGT CGT CGT GTT CGT CGT GAA CGT GCT CGT GCT CAC CCG TGC TGC CGT CCG ACT GCT TAC GAA GAC GAA GTT TCT TTC-3' (SEQ ID NO:20); 5'-CGG ATC CTT AAA CGC AAG CGC ATT CAC GAG CAG ACA GTT CCT GCA GAG TGT GGT AAC GAG AGT GAA CGT CCA GGA AAG AAA CTT CG-3' (SEQ ID NO:21). These oligos were gel purified and then annealed for 10 min at 680 C followed by 30 min at 22 C to form a linear sequence. The annealed oligos were extended with Klenow fragment, kinased and ligated into the Bluescript KS plasmid. After verifying the authenticity of the cloned NTN fragment by DNA sequencing, the fragment was transferred to the NdeI and BamHI site of the expression vector pET30a(+) (Novagen, Madison, WI). A histidine tag followed by a enterokinase site was placed at the amino terminus of the NTN sequence by inserting oignuceide linkers A (5'-TAT GCA CCA TCA TCA TCA TCA TGA CGA CGA CGA CAA GGC-3')(SEQ ID NO:2 2 and B (5'-TAG CCT TGT CGT

CGT

CGT CAT GAT GAT GAT GAT GGT GCA-3')(SEQ ID NO:23) into the Ndel site.

The mature rat GDNF coding sequence was obtained from an embryonic-day-21 rat kidney cDNA library by PCR using primers 5'-CAG CAT ATG TCA CCA GAT AAA CAA GCG

GCG

GCA CT-3' (SEQ ID NO:2 4 and 5'-CAG GGA TCC GGG TCA

GAT

ACA TCC ACA CCG TTT AGC-3' (SEQ ID N0:2 5 The amplified cDNA fragment was subcloned into the NdeI and Sall sites of pET30a(+) A six-His tag and enterokinase site were added to the amino terminus of the NTN sequence at the Ndel site using linkers A and B as above.

The NTN and GDNF pET30a(+) constructs were sequenced to confirm their authenticity and then transformed into E. coni strain BL21/DE3. The transformed bacteria were grown at 370 C in 2XYT medium (30 g/ml kanamycin) with vigorous shaking. For GDNF production, IPTG was added to a final concentration of 1.0 mM to induce expression of the protein after the culture reached an optical density of A 600 0.7. Incubation was continued for an additional WO 98/46622 PCTIUS98/07996 46 2 h. Bacteria containing the NTN expression construct were grown for 24 h without IPTG.

Cells were harvested by centrifugation at 4000 X g for 20 min, solubilized with Buffer A (6 M guanidine HC1, 0.1 M Na phosphate, 10 mM Tris HC1, pH 8.0) at 1/10th volume of the original culture volume and rocked overnight. Lysate was centrifuged at 10,000 x g for min at 4 C. The supernatant was exposed to 4 ml of Nickel-NTA (nitrilo-tri-acetic acid) resin (Qiagen, Chatsworth, CA) per 1 liter original culture and washed with 10-20 column volumes of Buffer A, 10 column volumes of Buffer B (8 M urea, 0.1 M Na phosphate, 10 mM Tris HC1, pH and 5-10 volumes of Buffer C (8 M urea, 0.1 M Na phosphate, 10 mM Tris HC1, pH 6.3) until the A280 was <0.01. The recombinant NTN or GDNF protein was eluted with 10-20 ml Buffer E (8 M urea, 0.1 Na phosphate, 10 mM Tris, pH Fractions were collected and analyzed by SDS-PAGE. The eluate was immediately diluted to 50 ng/pl in Buffer B and dialyzed against 4 M Renaturation Buffer (4 M urea, 5 mM cysteine, 0.02% Tween 10% glycerol, 10 mM Tris HC1, 150 mM NaC1, 100 mM Na phosphate, pH 8.3, under argon) at 40 C overnight and then against 2 M Renaturation Buffer (as above except with 2 M urea) 2-3 days with changes every 24 h.

Ret phosphorylation assays, PI-PLC treatment TrnRl- and TrnR2-expressing fibroblasts were grown to confluence in DMEM plus 10% calf serum, treated with ng/ml recombinant NTN or GDNF for 10 min, or left untreated, and then lysed. A portion of the lysates was removed and assayed for total Ret expression by western blot analysis using an anti-Ret antibody. The remaining lysates were immunoprecipitated with an antiphosphotyrosine antibody and analyzed by western blot using the anti-Ret antibody. The results are shown in Figure 3A.

Anti-Ret immunoblot analysis for total Ret shows WO 98/46622 PCT/US98/07996 47 that expression by the Ret and Ret/TrnR2 expressing clones of the immature (150kD) intracellular Ret protein and the glycosylated mature Ret protein (170 kD) was essentially the same whether treated with NTN or GDNF or left untreated (Total, Fig. 3A).

However, when the lysates were immunoprecipitated with anti-phosphotyrosine antibody before anti-Ret western analysis (IP, Fig. 3A), no Ret protein was observed in lysates from fibroblasts expressing only the Ret tyrosine kinase (Ret, Fig. 3A) whether left untreated or treated with NTN or GDNF at doses ranging from 50 to 3000 ng/ml (Fig. 3A and data not shown). A tyrosinephosphorylated band of approximately 170 kD was observed only in cells which expressed both Ret and TrnR2 and which were treated with GDNF or NTN, indicating that TRNinduced activation of the mature Ret protein required the presence of both Ret and TrnR2 (IP, Fig. 3A). Similar results were seen for Ret/TrnRl expressing cells (data not shown).

Further evidence that the effects of both GDNF and NTN could be mediated by TrnR2 was obtained by treating fibroblasts expressing Ret/TrnR2 with 1 U/ml PI-PLC for 45-60 min at 37°C and then washing the PI-PLC treated cells prior to NTN or GDNF treatment. As shown in Fig.

2B, PI-PLC treatment, which specifically cleaves GPIlinked proteins from the cell surface, significantly depleted the NTN or GDNF induced phosphorylation of Ret (compare lanes with lanes). These data indicate that TrnR2, a putative GPI-linked protein, can form a functional receptor with Ret for NTN or GDNF. This is analogous to the previously described requirement of TrnRl as a co-receptor with Ret for GDNF signaling (Treanor et al., supra; Jing et al., supra).

To further characterize the activities of these two TRN co-receptors, the effects of 0 to 100 ng/ml of NTN or GDNF on Ret phosphorylation was investigated. As shown WO 98/46622 PCT/US98/07996 48 in Fig. 3C, the Ret stimulating activity of both coreceptors is dose dependent since more phosphorylation of Ret in either construct was observed at higher levels of GNDF and NTN. However, while the extent of Ret phosphorylation in Ret/TrnRl expressing fibroblasts treated with NTN or GDNF was approximately equivalent at all doses tested, the TrnR2/Ret expressing fibroblasts were more sensitive to NTN treatment than GDNF treatment.

In particular, Ret phosphorylation was clearly observed in response to NTN treatment at 0.3 ng/ml; an equivalent response to GDNF was observed at 10 ng/ml. Similar results were obtained with multiple batches of recombinant GDNF and NTN, and with another stable TrnR2/Ret transfectant (data not shown).

The observed difference in the dose-response curves of the Ret/TrnRl and Ret/TrnR2 fibroblasts to NTN and GDNF suggests that there is a difference in the functional affinity of the ligands for the two receptor complexes, TrnRl/Ret and TrnR2/Ret. The TrnR2/Ret complex may function preferentially as a NTN receptor, whereas the TrnRl/Ret complex responds equivalently to either factor.

Example 2 This example illustrates that the short splice variant of TrnR2 (TrnR2-SV) can mediate signal transduction through Ret like the long splice variant (TrnR2-LV).

3T3 fibroblasts coexpressing Ret and either TrnR2-LV or TrnR2-SV were generated by cotransfecting a clonal Ret-expressing 3T3 cell line with a SV2-His plasmid and a cDNA encoding TrnR2-LV and then selecting the desired transfectants in 2 mM L-histidinol essentially as described in Example 1.

The recombinant fibroblasts were stimulated with either GDNF, neurturin or persephin at 100 ng/ml for minutes and then lysed. To determine the amount of WO 98/46622 PCT/US98/07996 49 active phopshorylated Ret after stimulation with each growth factor, the lysates were immunoprecipitated using an anti-phosphotyrosine antibody, and then analyzed by western blot using an anti-Ret antibody as described in Example 1. The results are shown in Fig. 3D.

When stimulated with GDNF or neurturin (NTN), the amount of tyrosine-phosphorylated Ret was approximately equal in cells coexpressing TrnR2-SV as that in cells coexpressing TrnR2-LV. In addition, neurturin stimulation produced more Ret phosphorylation than GDNF stimulation in both the TrnR2-SV-expressing and TrnR2-LVexpressing cells, which is consistent with the hypothesis that the TrnR2-LV/Ret complex has a preferential affinity for neurturin over GDNF.

These data indicate that the short and long splice variants of TrnR2 are essentially equivalent in mediating GNDF and neurturin signaling through Ret activation.

Thus, it is believed the short splice variant contains all the structural elements necessary for binding to GDNF and neurturin and presenting these ligands to Ret in the appropriate orientation such that Ret is phosphorylated.

Neither cell line responded to persephin (PSP), indicating that this family member acts through a differenct co-receptor complexed with Ret or through a different receptor complex altoghether.

Example 2A This example illustrates that a soluble TrnR2 polypeptide specifically binds to the GNDF and NTN members of the TRN family.

A cDNA encoding a soluble TrnR2 receptorimmunoglobulin fusion protein was prepared by fusing a polynucleotide encoding the amino acid sequence from methionine at position -21 through glycine at position 411 of SEQ ID NO:2, nucleotides 36-1331 of SEQ ID NO:1, to a polynucleotide encoding the Fc region of human IgG, using the pIgPlus vector system (Invitrogen, WO 98/46622 PCT/US98/07996 Carlsbad, CA). The resulting construct was transfected into COS cells and stable clones were selected using 1 mg/ml G418. Stable COS clones were grown in conditioned medium from which the secreted TrnR2-Fc fusion protein was purified using protein-A chromotography.

This soluble TrnR2-Fc fusion protein was then used in an ELISA binding assay to determine if soluble TrnR2- LV is capable of binding to GDNF, NTN and PSP. Solutions containing 250 ng/ml of GDNF, NTN or PSP were prepared and 50 pl of each solution was applied to separate Maxisorb ELISA plates (Nunc) 12.5 ng growth factor per well) and the growth factor was allowed to bind for 1 hr at room temperature. The wells of each plate were then washed, blocked, and incubated with increasing amounts of the soluble TrnR2-Fc fusion protein. The wells were washed again and bound TrnR2-Fc protein was detected using an anti-human IgG-HRP conjugated secondary antibody (Jackson Immunoresearch), and TMB liquid substrated detection reagent (Sigma). The amount of luminescence was plotted against the amount of TrnR2-Fc protein used in the assay and the results are shown in Fig. 3(E).

In this immunoassay, approximately equal amounts of soluble TrnR2-Fc bound to both GDNF and NTN when the fusion protein was added at less than 1 nM. However, at larger amounts of added fusion protein, much larger amounts of soluble TrnR2 bound to immobilized NTN than to immobilized GDNF, demonstrating that soluble TrnR2 also has greater affinity for NTN than GDNF as was shown in Example 1 for membrane bound TrnR2.

The specificity of TrnR2 for NTN and GDNF is indicated by the lack of its binding to PSP at all amounts of receptor tested.

Example 3 This example illustrates the expression of TrnRl and TrnR2 in various tissues.

WO 98/46622 PCT/US98/07996 51 TrnR2 expression in adult mouse is more limited than TrnRl expression TrnR2 expression in adult mouse was investigated by Northern blot analysis of total RNA (25 fg) isolated from various adult mouse.tissues and electrophoresed in a 1% agarose/formaldehyde gel and blotted onto a nylon membrane (Zetaprobe) using standard procedures (Chomczynski and Sacchi, Annal. Biochem 162: 156-159, 1987, incorporated herein by reference). To verify that equal amounts of total RNA were present in each lane, the 28S ribosomal RNA band was visualized by staining with ethidium bromide. RNA homologous to TrnR2 was detected by probing the blot with a 32 P-labeled fragment of TrnR2 cDNA. The results are shown in Figure 4.

RNA hybridizing to the TrnR2 probe was observed in brain and testis. Two messages were observed in brain, differing only slightly in size. These two bands likely correspond to the two splice forms found in brain while performing RACE PCR to amplify the 5' end of the TrnR2 cDNA, the shorter of which is missing 399 nucleotides from the coding region (see Fig. Two different bands were also observed in testis, which were significantly smaller kb) than either of the transcripts detected in the brain kb). One of the smaller TrnR2 messages in testis may be analogous to a small TrnRl mRNA reported which encodes a truncated protein of 158 amino acids (Treanor et al., 1996). Low-level expression may also be present in the spleen and in the adrenal.

These results indicate that the tissue distribution of TrnR2 is more limited than TrnRl in the adult animal, which has been detected in liver, kidney, and brain of adult rat and mouse (Jing et al., supra).

Analysis of TrnRl, TrnR2. and Ret expression in targets of GDNF and NTN Comparison of TrnR2 and TrnRl expression was also investigated in known sites of GDNF and/or NTN action by WO 98/46622 PCT/US98/07996 52 in situ hybridization analysis. Mouse tissue samples were obtained and prepared for in situ hybridization as described previously Wanaka et al., Neruron 5: 267-281, 1990, which is incorporated herein by reference). The results of in situ hybridization of these fresh frozen tissue samples with antisense 33 P-labeled RNA probes transcribed from fragments of TrnRl, TrnR2 and Ret cDNAs are shown in Figure In situ hybridization analysis showed only low-level expression of TrnR2 in the substantia nigra in the adult mouse, and in the ventral mesencephalon of an E14 mouse, in contrast to high-level expression of TrnRl and Ret (Fig. 5A and data not shown). Motor neurons in the ventral horn (vh) of the adult spinal cord also express TrnRl and Ret, but not TrnR2 (Fig. 5B). Ret is localized predominately to motor neurons, whereas TrnRl shows additional staining in the intermediate and dorsal horns of the cord. TrnR2 is highly expressed in the developing and adult dorsal root ganglia (drg), along with Ret and TrnRl (Fig. 5C and data not shown). In addition, strong expression of TrnRl and TrnR2, but not Ret, was observed in the exiting nerve root (Fig. 5D). In the developing kidney and gut there is high level expression of TrnRl and Ret, but not TrnR2 (Fig. Finally, significant expression of both TrnR2 and Ret was observed in the rat superior cervical ganglion (SCG), with only low-level, diffuse staining of TrnRl (Fig. These data indicate a partially overlapping expression pattern for TrnRl and TrnR2 in embryonic and adult central and peripheral nervous tissue. In several areas of known GDNF action, including nigral and motor neurons, high levels of TrnR1 and Ret are expressed, with only low or undetectable levels of TrnR2 expression.

Based on this initial survey, TrnR2 expression is largely limited to neuronal tissue in both embryo and adult, with highest levels of expression in sensory and sympathetic WO 98/46622 PCT/US98/07996 53 neuronal populations.

Example 4 This example illustrates that both NTN and GDNF promote the survival of newborn rat SCG neurons in culture through their activation of the Ret signaling pathway and this activation is likely mediated by TrnR2, not TrnRl.

Neuronal cultures were prepared from the SCG of postnatal day-i rats using known procedures (Martin et al., J. Cell Biol. 106:829-844, incorporated herein by reference). The SCG cultures were plated on collagencoated dishes and maintained in NGF for seven days and then deprived of NGF by switching to medium lacking NGF and containing an anti-NGF antibody (Ruit et al., Neuron 8: 573-587, 1992, incorporated herein by referencer.

After 2-4 h, this medium was replaced with medium containing 50 ng/ml NTN, GDNF, or NGF. After 10 min incubation in these growth factors, lysates were prepared and assayed for total Ret protein and tyrosinephosphorylated Ret as described in Example 1. Tyrosine phosphorylated Ret protein was observed in cells treated with NTN but not in NGF-treated cells (data not shown).

Thus, both NTN and GDNF can activate the Ret receptor.

It had been shown that the survival-promoting ability of GDNF on several neuronal populations including SCG neurons was significantly reduced by PI-PLC treatment (Treanor et al., supra). To assess whether NTN-induced Ret activation is similarly affected, SCG cultures were grown in serum-free N2 medium to maximize the activity of PI-PLC and then treated with 1 U/ml PI-PLC prior to the addition of NTN (50 ng/ml). NTN-induced tyrosine phosphorylation of Ret was significantly reduced in cultures treated with PI-PLC (data not shown). Thus, activation of the Ret PTK by NTN and GDNF appears to be mediated by a GPI-linked protein.

As shown in Figure 5D, in situ hybridization WO 98/46622 PCT/US98/07996 54 analysis indicated that TrnR2 and Ret are expressed at high levels in rat SCG neurons, whereas TrnRl is expressed diffusely and does not appear to be localized to neurons. To further assess the cellular localization of TrnRl, TrnR2 and Ret mRNAs in this ganglion, their expression in primary SCG cultures was analyzed. Because the primary cultures contain a small contaminating population of non-neuronal cells (Schwann cells and fibroblasts), neuronal specific messages can be identified by inducing apoptosis in the neuronal population. Removal of NGF from the culture medium results in near complete death of the neuronal population within 48 hours (Martin et al., J. Cell Biol. 106:829- 844, 1988; Deckwerth et al., J. Cell Biol. 123:1207-1222, 1993; Edwards et al., J. Cell Biol. 124:537-546, 1994, which are incorporated herein by reference). During this period, neuronal messages decrease whereas messages from non-neuronal cells remain constant (Freeman et al., Neuron 12:343-355, 1994; Estus et al., J. Cell Biol.

127:1717-1727, 1994, each incorporated herein by reference).

The amount of TrnRl, TrnR2, Ret mRNA in neuronal cultures was assessed using semiquantitative reverse transcription-PCR (RT-PCR) as described in Freeman et al., supra and Estus et al., supra. Five day SCG cultures were switched to medium containing anti-NGF antibodies for various times. Polyadenylated RNA was isolated from the cultures at 0, 6, 12, 18, 24 and 36 hours after NGF removal using the QuickPrep Micro Kit (Pharmacia, Piscataway NJ) according to the manufacturer's instructions. Half of the poly-A RNA was converted to cDNA by reverse transcription with Moloney murine leukemia virus reverse transcriptase with random hexamers (16 gM) as primers. cDNA from approximately 150 cells was used in a 50 yl PCR reaction using the following primer sets: mouse Ret forward WO 98/46622 PCT/US98/07996 TGGCACACCTCTGCTCTATG-3' (SEQ ID NO:26) and reverse TGTTCCCAGGAACTGTGGTC-3' (SEQ ID NO:27); TrnRl forward GCACAGCTACGGGATGCTCTTCTG-3' (SEQ ID NO:28) and reverse 5'-GTAGTTGGGAGTCATGACTGTGCCAATC-3' (SEQ ID NO:29); TrnR2 forward 5'-AGCCGACGGTGTGGCTCTGCTGG-3' (SEQ ID NO:30) and reverse 5'-CCAGTGTCATCACCACCTGCACG-3' (SEQ ID NO:31).

After amplification, the PCR products were separated by electrophoresis on 10% polyacrylamide gels, visualized by autoradiography of the dried gels, and quantified with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale CA).

The results are shown in Figure 6.

Ret and TrnR2 messages decreased as the neurons died, in a manner similar to neuron-specific enolase (NSE). In contrast, TrnRl levels remained constant, similar to the Schwann cell marker S-100.

These data indicate that Ret and TrnR2 expression is largely limited to neurons in neonatal rat SCG cultures, and likely mediates the functional response of these neurons to NTN and GDNF. This is consistent with in situ hybridization analysis (Fig. 5D) which also indicates that the expressed receptor complex in SCG neurons consists of TrnR2 and Ret. Interestingly, NTN is more potent in promoting the survival of SCG neurons than GDNF (Kotzbauer et al., supra), which is consistent with the higher sensitivity of the TrnR2/Ret receptor complex to NTN treatment (Fig. 3C). Although some low level neuronal expression of TrnRl cannot be excluded by this assay, these data indicate TrnRl is predominantly expressed in the non-neuronal population, consistent with its previously observed expression in Schwann cells (Treanor et al., supra).

Example This example illustrates that TrnRl, but not TrnR2, is up-regulated in distal sciatic nerve after nerve injury.

GDNF is a well characterized trophic factor for both WO 98/46622 PCT/US98/07996 56 embryonic and adult motor neurons (Henderson et al., Science 266:1062-1064, 1994; Yan et al., Nature 373:341- 344, 1995; Oppenheim et al., Nature 373:344-346, 1995; Li et al., Proc.Natl.Acad.Sci.USA 92:9771-9775, 1995, all are incorporated herein by reference). In the adult animal, GDNF expression is up-regulated in the distal segment of the sciatic nerve after transection, and in denervated muscle (Trupp et al., J. Cell.Biol. 130:137- 148, 1995; Springer et al., Exp.Neurol. 131:47-52, 1995, each is incorporated herein by reference). This is similar to observations regarding NGF and the p75 low affinity neurotrophin receptor (p75NTR), which are both upregulated by Schwann cells in the distal segment of the sciatic nerve after transection (Taniuchi et al., Proc.Natl.Acad.Sci.USA 83:4094-4098, 1986; Heumann et al., J.Cell.Biol. 104:1623-1631, 1987, each is incorporated herein by reference). Because GDNF is upregulated after injury, and because TrnRl is expressed by Schwann cells (Treanor et al., supra), the expression of TrnRl and TrnR2 in the distal segment of the rat sciatic nerve before and after transection was examined to determine if one or both of the TRN co-receptors is upregulated after transection.

Northern blot analysis was performed on total RNA isolated from normal rat sciatic nerve and from the distal segment of the sciatic nerve seven days posttransection using 32 P-labeled TrnRl and TrnR2 cDNA fragments as probes. Rat sciatic nerves were transected and recovered as previously described (Araki et al., Neuron 17:353-361, 1996, incorporated herein by reference). Brain total RNA was included on the blot as a positive control for detection of TrnR2 mRNA. The results are shown in Figure 7.

Seven days after nerve transection the distal portion of the sciatic nerve showed a dramatic increase in the level of TrnRl mRNA from the level observed before WO 98/46622 PCT/US98/07996 57 transection In contrast, TrnR2 mRNA was not detected in the nerve either before or after transection The 28S ribosomal RNA band, visualized using ethidium bromide, is shown below to demonstrate equal loading of total RNA samples.

Consistent with the observed differential expression of TrnRl and TrnR2 in Schwann cells, RT-PCR analysis of the JS-1 Schwann cell line also showed expression of TrnRl, but not TrnR2 (data not shown). These results indicate that TrnR2 is unlikely to play a major role in Schwann cell mediated peripheral trophic support of the regenerating nerve. However TrnRl, in conjunction with GDNF produced by the distal sciatic nerve and muscle, could potentially provide a potent trophic substrate for growth of the regenerating nerve.

Example This example illustrates additional analysis by in situ hybridization of the expression of neurturin, GDNF and their receptors in the central nervous system of the adult mouse.

MATERIALS AND METHODS Riboprobes Riboprobes were synthesized from plasmids containing mouse cDNA sequences of neurturin (nucleotides 293-598, 441-675 of GenBank accession number U78109), GDNF (nucleotides 256-935 of GenBank accession number D88264), GFRa-l (TrnR1, nucleotides 574-1069 of GenBank accession number U59486), GFRa-2 (TrnR2, nucleotides 1-569, 1002-1417 of GenBank accession number AF002701) and Ret (nucleotides 207-611 of GenBank accession number X67812).

Both GFRa-2 (TrnR2) probes contained sequences that are present in both splice variants of TrnR2 mRNA (Baloh et al., Neuron 18:793-802, 1997). The Ret probe also included sequence present in all of the known ret splice variants. Plasmids were linearized with appropriate restriction enzymes and transcribed in vitro with 50 pCi WO 98/46622 PCT/US98/07996 58 of UTP (NEN Dupont) by using T3, T7, or SP6 RNA polymerase. All experiments were performed within 24 hours of probe synthesis. The probes were clearly specific based on the distinct expression patterns observed for the different mRNA. Two different sense probes were also used to ensure specificity further.

Preparation of Tissue Animals were housed and treated in accordance with the guidelines of NIH and Washington University. Young adult (6-8 weeks) female ICR mice that were at mid to late gestation or recently delivered were anesthetized with an overdose of xylazine (20 mg/ml): ketamine (100 mg/ml): acepromazine (10 mg/ml), Brains were removed, frozen on dry ice, and sectioned at 14 ym in the sagittal or coronal plane on a cryostat. Frozen sections were thawed then postfixed in 4% paraformaldehyde in PBS, pH 7.4, for 20 minutes.

Sections were pretreated for hybridization as follows: minutes in PBS; 3 minutes in 0.2% glycine in PBS; minutes in PBS; 10 minutes in 0.1M triethanolamine, pH 8; 2x10 minutes in 0.025% acetic anhydride/0.1M triethanolamine; and, 5 minutes in PBS. Sections were then dehydrated in a graded series of alcohol and defatted with chloroform. 3P-labeled RNA probes were diluted in hybridization buffer (50% formamide, Tris-HCL, pH 7.5, 5mM EDTA, lx Denhardt's solution, dextran sulfate) to lx10 6 counts per 75 pl. Slides were incubated overnight at 55*C in a humidified chamber with 75y1 of hybridization solution per slide. After hybridization, slides were washed as follows: 4x15 minutes in 4x SSC at room temperature; 20 minutes in 2xSSC at 55"C; 2x15 minutes in RNase buffer (0.5M NaCI, Tris-HC1, pH 8, ImM EDTA) at 370C; 30 minutes in pg/ml RNase A in RNase buffer at 37"C; 2x15 minutes in RNase buffer at 37*C; 20 minutes in 2xSSC at 55°C; minutes in 1xSSC at 65*C; and, 30 minutes in 0.1x SSC at WO 98/46622 PCT/US98/07996 59 Slides were then dehydrated and dipped in Kodak NTB2 emulsion. After an exposure time of 14-18 days, slides were developed and counter-stained with hematoxylin and eosin. Neurons were identified based on morphology. Cells with a pale-staining cytoplasm and large-round, pale nuclei were identified as neurons.

Cells with small oval or irregularly shaped, darkstaining nuclei were identified as glial cells. Slides were photographed and figures were prepared from the photographs with Adobe Photoshop 4.0.1. With the exception of dust removal, and dodging/burning to produce uniform tone photographs were not altered.

Labeling was considered specific if it was above the level of background. Background label is defined as labeling obtained with a sense probe or labeling that was not specifically localized to cells. The intensity of labeling for each probe was classified as follows: high-the greatest intensity of labeling observed for a particular probe; moderate-high-to-medium level of labeling; low-easily detected but low level of intensity; barely detected-very low level of labeling in few or scattered cells; or, not detected-no labeling above background. Abbreviations are according to Paxinos and Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press Inc., 1986; and Franklin and Paxinos, The Mouse Brain in Stereotaxic Coordinates, Academic Press Inc., 1997.

RESULTS

Forebrain In the forebrain, GFRa-1 (TrnRl), GFRa-2 (TrnR2), and Ret mRNAs were widely expressed in the septum and olfactory system (Figures 8A-C; Table GFRa-1 (TrnRl), GFRa-2 (TrnR2), and Ret mRNAs were expressed in the medial and lateral septal nuclei, the nucleus of the diagonal band of Broca, and the piriform cortex (Figures 8A, 8B). In each of these areas, the receptors were WO 98/46622 PCT/US98/07996 expressed in cells morphologically consistent with neurons. All three receptor components were expressed at higher levels in the lateral septal nucleus than in the medial septal nucleus. Ret was expressed at much lower levels than either GFRa-1 (TrnRl) or GFRa-2 (TrnR2) in the lateral septal nucleus. In the nucleus of the diagonal band, all three receptors were expressed at low levels with GFRa-2 (TrnR2) expressed at the lowest level.

GFRa-1 (TrnRl) and GFRa-2 (TrnR2) mRNAs were expressed in the absence of detectable Ret mRNA in the neocortex, claustrum, endopiriform nucleus, the bed nucleus of the stria terminalis, the basal nucleus of Meynert, the ventral pallidum, and the olfactory tubercle (Figure 8, Table GFRa-1 (TrnRl) was expressed at high levels in scattered clusters of neurons in the ventral pallidum.

GFRa-2 (TrnR2) was expressed in neurons in the ventral pallidum at much lower levels and in fewer cells than GFRa-1 (TrnRl).

NTN (Figures-8D, 9D) and GDNF (data not shown) mRNAs were expressed in piriform cortex, in the hippocampus and, at low levels, in neocortex (Figures 8D, 9D; Table In addition, expression of GDNF was seen at moderate levels in the olfactory tubercle and at very low levels in the nucleus accumbens, the globus pallidus, the ventral pallidum, the subiculum, and the striatum (Table 2).

Olfactory Bulb In the olfactory bulb, mRNAs for all three receptors were expressed in the glomerular layer and in the granule layer (Table In the glomerular layer, GFRa-l (TrnRl) and Ret were expressed at moderate levels and GFRa-2 (TrnR2) was expressed at a lower level. In the granule layer, GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were expressed at higher levels than Ret. In the mitral cell layer, both GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were highly expressed in the absence of ret. GFRa-1 (TrnRl) was WO 98/46622 PCT/US98/07996 61 expressed in the absence of Ret and GFRa-2 (TrnR2) in the internal plexiform layer and the external plexiform layer.

NTN and GDNF mRNA expression were not detected in the olfactory bulb (Table 2).

Midbrain Receptors for NTN and GDNF were widely expressed in the midbrain. Most notably, mRNAs for all three receptor components were detected in the pars compacta of the substantia nigra, in which dopaminergic neurons, responsive to both GDNF and NTN in adult mice are located (Figure 10; Table 2) (Lin et al., 1993; Rosenblad et al., Soc. Neurosci. Abstr. 23:248.12, 1997). Receptor expression in the substantia nigra was predominantly seen in the pars compacta with much sparser labeling in the pars reticulata. In particular, Ret and GFRa-1 (TrnRl) were expressed at highest levels, while GFRa-2 (TrnR2) was expressed at substantially lower levels. All three receptor components were predominantly, but not exclusively, expressed in neurons. Expression of all three receptor components was also found in the ventral tegmental area (VTA), the periaqueductal grey (PAG), the rostral linear raphe (RLi), the interfascicular nucleus and the Edinger Wesphal nucleus All three receptor components were also expressed in the large motor neurons of the oculomotor nucleus GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were expressed in the absence of Ret in the superficial layers of the superior colliculus, in the interpeduncular nucleus, and in the cerebral cortex (Figure 10). In addition, GFRa-1 (TrnRl) and Ret, but not GFRa-2 (TrnR2), were expressed in the red nucleus (Table 2).

NTN mRNA was expressed at a barely detectable level in the oculomotor nucleus. NTN expression was not seen in any other areas of the midbrain. GDNF mRNA was expressed at very low levels in the substantia nigra pars WO 98/46622 PCT/US98/07996 62 compacta, in the superficial layers of the superior colliculus and in the interfascicular nucleus.

Thalamus Receptor components were expressed in several nuclei in the thalamus including both relay and association nuclei (Figures 9A-C; Table The receptors were most prominently expressed in the reticular nucleus the zona incerta, and the habenular nuclei (Figures 9A-C).

In the reticular nucleus, mRNAs for GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were expressed at high levels while Ret mRNA was expressed at moderate-to-low levels. In the habenular nuclei, GFRa-1 (TrnRl) was expressed at high levels and the other two receptors were expressed at lower levels, particularly in the lateral habenular nucleus. All three receptor components were also expressed at low levels in the medial geniculate nucleus (Figure Several of the sensory and motor relay nuclei expressed Ret mRNA at very low levels in the absence of GFRa-1 or GFRa-2 (Figure 9; Table These included the two main sensory relay nuclei, the ventroposteromedial (VPM) and the ventroposterolateral (VPL), and the primary motor relays, the ventromedial (VM) and ventrolateral (VL) nuclei. In the other major sensory relay, the posterior nuclear group both Ret and GFRa-1 (TrnR1) were expressed at low levels but GFRa-2 (TrnR2) was not detected. The same pattern was observed in the laterodorsal and lateroposterior nuclei, and the mediodorsal nucleus in which Ret and GFRa-1 (TrnRl) were expressed at low levels and GFRa-2 (TrnR2) was absent.

In addition, Ret and GFRa-2 (TrnR2) were expressed in the absence of GFRa-1 (TrnRl) in the subthalamic nucleus (Figures 9A-C).

NTN was expressed at moderate levels in the anteromedial and anteroventral nuclei of the thalamus (Figure 9D). GDNF mRNA was expressed at low levels in WO 98/46622 PCT/US98/07996 63 the anteromedial and anteroventral nuclei and, at very low levels in the reticular, ventromedial, ventrolateral, ventroposteromedial, and ventroposterolateral nuclei, and in the posterior nuclear group and the zona incerta (Table 2).

Hypothalamus In the hypothalamus, GF (TRN) receptor components were prominently expressed in many areas (Figure 9; Table All three receptor components were detected in the periventricular nucleus, the medial preoptic nuclei, both central and median, in the medial and lateral preoptic area, the ventromedial (VM) and dorsomedial (DM) hypothalamic nuclei, the supramammillary nucleus, and the posterior, anterior and lateral hypothalamic areas.

GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were expressed, in the absence of Ret, in the paraventricular nucleus and the arcuate nucleus.

NTN was expressed in the supraoptic, and paraventricular nuclei (Figure 11A). NTN expression in the supraoptic and paraventricular nuclei was particularly intense. In the supraoptic and paraventricular nuclei, NTN expression was localized to cells whose morphology is consistent with that of the large secretory neurons that are found in these nuclei (Figures 11B, 11C). GDNF mRNA was expressed at barely detectable levels in the hypothalamus in the dorsomedial, ventromedial, arcuate and medial mammillary nuclei (Table 2).

Brainstem GF (TRN) receptor components were expressed in a number of brainstem nuclei including cranial nerve nuclei (Figure 12; Table All three receptor components were expressed in the facial motor nucleus, in the region of the nucleus ambiguous, the abducens nucleus, the prepositus hypoglossal nucleus, the spinal trigeminal sensory nuclei, the vagal dorsal motor nucleus, the WO 98/46622 PCT/US98/07996 64 lateral and medial vestibular nuclei, and the dorsal and ventral cochlear nuclei. GFRa-1 (TrnRl) and Ret were also expressed, in the absence of GFRa-2, in the region of the inferior salvitory nucleus and nucleus retroambiguous, and in the hypoglossal nucleus, and in motor neurons in the trigeminal motor nucleus. In the principal sensory trigeminal nucleus, Ret was expressed in the absence of GFRa-1 TrnRl) and GFRa-2 (TrnR2). In the cranial motor nuclei, the receptors were expressed in large motor neurons.

Other areas of the brainstem in which expression of all three receptors was detected included the raphe nuclei, the inferior colliculus, the pontine reticular nucleus, the gigantocellular reticular nucleus, the tegmental nuclei, and the locus coeruleus.

NTN was expressed at very low levels in the region of the nucleus ambiguous. GDNF mRNA was expressed at a low level in the facial motor nucleus and the ventral cochlear nucleus (Figure 12C).

Spinal Cord Transverse sections of cervical, thoracic and lumbar spinal cord were examined. The labeling pattern for each of the receptors was the same at each level studied.

GFRa-1 (TrmRl) and Ret were expressed prominently in motor neurons of the ventral horn (Figures 13A, 13C).

Low levels of GFRa-2 (TrnR2) were expressed diffusely in the spinal cord gray matter with highest levels of expression found in the superficial layers of the dorsal horn (Figure 13B). In the ventral horn GFRa-2 (TrnR2) mRNA was expressed in a few scattered motor neurons.

Cerebellum In the cerebellum, Ret and GFRa-1 (TrnRl) were expressed at a high level in the Purkinje cell layer (Figures 14A, 14C). Both Ret and GRFa-1 (TrnRl) were expressed in cells adjacent to Purkinje cells, but not in Purkinje cells. GFRa-2 (TrnR2) was strongly expressed in WO 98/46622 PCT/US98/07996 Purkinje cells in a pattern complementary to Ret and GFRa-1 (TrnRl) expression (Figure 14B). GFRa-2 was also expressed at a low level in the granule cell layer and Ret was expressed at a low level in the molecular layer.

In the deep cerebellar nuclei, all three receptor components were detected, with Ret and GFRa-1 (TrnRl) detected at higher levels than GFRa-2 (TrnR2) (Table 2).

Both NTN (Figure 14D) and GDNF (Table 2) were expressed at low levels in the Purkinje cell and granular layers of the cerebellum. NTN and GDNF were not detected in the molecular layer. NTN, but not GDNF, was detected in the deep cerebellar nuclei.

WO 98/46622 PCT/US98/07996 TABLE I -Sumimary of GF Factor and Receptor Expression NTN GDNF GFRcx-1 GFRax-2

RET

FOREBRAIN

Olfactory Bulb Glomerular Layer Mitral Cell Layer Internal Plexiform Layer External Plexiform Layer Granule Layer Olfactory Tubercle Piriformn Cortex Claustrum Dorsal Endopiriform Nucleus Medial Septal Nucleus Lateral Septal Nucleus Nucleus Diagonal Band Bed Nucleus Stria Terminalis Nucleus Accumbens: Amygdala Basal Nucleus of Meynert Ventral Pallidumn Globus Pallidus Striatumn

MIDBRAIN

Substantia Nigra Compacta Reticulata Ventral Tegmental Area Periaqueductal Gray Rostral Linear Raphe Dorsal Raphe Oculomotor Nucleus Edinger' Westphal Superior Colliculus Red Nucleus Interfascicular Nucleus Interpeduncular Nucleus

CORTEX

Neocortex.

Cingulate Cortex Hippocampus: Subiculum

CAI

CA2 CA3 Dentate 14 14 14 14 14 +14 +14 1-H- 14 14 14 14 14 14 14 14 14 14 14 14 14 :1: +14 14+ +14 14 14

OB

Tu Pir Cl DEn

MS

LS

DB

BST

Acb

A

B

VP

GP

CPU

SN

SNc SNr

VTA

PAG

RLI

DR

3

EW

SC

R

IF

'P

1-H- 14 14 14 14 14 SUBSTITUTE SHEET (RULE 26) WO 98/46622 WO 9846622PCTIUS98/07996 67 TABLE 1'-Sumnmary 01 Factor and Receptor Expres, 5 ,on NTN GDNF GFRoa-i GFRcx-2 RET

CEREBELLUM

Granule Cell Layer Purkinje Cell Layer Molecular Layer Deep Nuclei

THALAMUS

Medial Habenula Lateral Habenula Reticular Nucleus Mediodorsal, Nucleus Anteromedial Nucleus Anteroventral Nucleus Laterodorsal Nucleus Lateral Posterior Nucleus Zona. Incerta Subthalamnic Nucleus Ventromedial Ventrolateral.

Ventral Anterior Posterior Nuclear Group Ventroposterolateral.

Ventroposteromedial Lateral Geniculate Medial Geniculate

HYPOTHALAMUS

Medial Preoptic, Nucleus Central Median Preoptic Area Medial Lateral Periventricular Nucleus Tubercinerium Lateral Hypothalamic Area Anterior Hypothalamic Area Posterior Hypothalamic Area Ventromedial. Hypothalamic Nucleus Dorsomedial. Nucleus Supraoptic Nucleus Arcuate Nucleus Medial Manimillary Nucleus Supramarmjillary Nucleus Paraventricular Nucleus H- -H- +4* H- H- 4* 4* 4* 4* 4* 4* -4* 4*

-H-

GR

P

Mo I MHb LHb Rt

MID

AM

AV

LD

LP

ZI

Smh

VM

VL

VA

Po

VPL

VPM

LGN

MGN

MP

4* 4* -1* 4* 4* Pe

-+T

ZE LHA

AHA

PHA

:b VM

DM

so Arc

MM

sum

PV

SUBSTITUTE SHEET (RULE 26) WO 98/46622 PCTIUS98/'07996 68 TABLE V -Sumnmary of GF Factor and Receptor Expression NTN GDNF GFRax-i GFR(X-2 RET

BRAINSTEM

Median Raphe Raphe Pallidus Raphe Magnus Inferior Colliculus Preolivary Region Trigemninal Principalis Spinal Trigemninal Motor Trigeminal Nucleus Ambiguous Nucleus Retroambiguous Vagal Dorsal Motor Nucleus Hypoglossal Prepositus Hypoglossal Abducens Facial Nucleus Dorsal Cochlear Nucleus Ventral Cochlear Lateral Vestibular Medial Vestibular Inferior Salvitory Pontine Reticular Nucleus Gigantocellular reticular Tegmental Nuclei Locus Coeruleus 4* 4* 4-4* 4* 4* 44+ 4* 4* +4* 4* 4* 44+ +44 4* 4* 4* MnR Rpa, RMg

[C

12 6 7

DC

VC

4* 4* 4* +4* 4* 4* Gi Ta

LC

SPINAL CORD Ventral Horn Dorsal Horn 4-4* VH 44 DH I Level of expression: I a, high; moderate; low; barely detected; not detected.

SUBSTITIUTE SHEET (RULE 26) WO 98/46622 PCT/US98/07996 69

DISCUSSION

In the experiments described in this example, we studied the expression of NTN and GDNF and their receptors in the adult mouse brain. GF (TRN) receptors are widely expressed throughout the adult brain. In many areas of the brain, including all the areas in which GFresponsive neurons are present, Ret and either GFRa-1 (TrnRl) or GFRa-2 (TrnR2) mRNA are present. In some areas of the brain, most notably the cerebral cortex and the hippocampus, co-receptors GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were expressed without Ret.

NTN and GDNF mRNA were expressed at lower levels and in fewer areas of the adult mouse brain than GF receptor (TrnR) mRNA. NTN and GDNF were expressed in several areas of the brain that receive projections from neurons expressing receptor mRNA. This expression pattern suggests that NTN and GDNF function through a classical target-derived mechanism of trophic factor action to maintain neuronal circuits in the mature CNS.

Neurons That Respond to NTN and/or GDNF in Adult Rodents Express GF (TRN) Receptors.

Several central neuronal populations respond to GDNF in the adult rodent including spinal motor neurons (Li et al., Proc. Natl. Acad. Sci. 92:9771-9775, 1995), facial motor neurons (Yan et al., Nature 373 (6512):341-344, 1995), dopaminergic neurons of the ventral midbrain (Bowenkamp et al., J. Comp. Neurol. 355:479-489, 1995; Kearns and Gash, Brain Res. 672:104-111, 1995; Tomac et al., Nature 373:335-339, 1995; Cass, J. Neurosci.

16(24):8132-8139, 1996; Bowenkamp et al., Exp. Neurol.

145:104-117, 1997; Choi-Lunberg et al., Science 275:838- 841, 1997; Lapchak et al., Neuroscl. 78(1):61-72, 1997), noradrenergic neurons of the locus coeruleus (Arenas et al., Neuron 15:1465-1473, 1995), and cholinergic neurons of the basal forebrain (Williams et al., J. Pharm. Exper.

Ther. 277:1140-1151, 1996). In addition, dopaminergic WO 98/46622 PCT/US98/07996 midbrain neurons are responsive to NTN in adult rats (Rosenblad et al., supra).

In the.experiments described in this example, we observed expression of GF (TRN) receptors in areas of the brain in which responsive neurons are located. For example, in vivo, GDNF is a potent neurotrophic factor for'spinal (Li et al., supra) and facial (Yan et al., supra) motoneurons in adult rodents. Both facial motor neurons and spinal motor neurons expressed Ret and GFRa-1 (TrnRl). In adult rodents, dopaminergic neurons in the pars compacta of the substantia nigra and in the VTA are protected from chemical and mechanical injury by NTN (Rosenblad et al., supra) or GDNF (Kearns and Gash, supra; Bowenkamp et al., 1997, supra). Ret, GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were all expressed in neurons in these areas of the ventral midbrain. All three receptor components were also expressed in the interfascicular nucleus and the rostral linear raphe. In adult mice, dopaminergic neurons in these nuclei are protected from MPTP (l-methyl-4-phenyl-l,2,3,6tetrahydropyridine) treatment by GDNF (Tomac et al., supra). GDNF treatment also protects cholinergic neurons of the septal/diagonal band nuclei from axotomy by fimbria/fornix section in adult rats (Williams et al., supra). In our experiments, we found GF (TRN) receptors in both the medial septal nucleus and in the nucleus of the diagonal band of Broca in neurons morphologically consistent with large cholinergic neurons characteristic of this area. In the locus coeruleus, GDNF treatment protects noradrenergic neurons from death (Arenas et al., supra). Neurons in the locus coeruleus expressed all three GF receptors. In summary, there is a strong positive coorelation between GF (TRN) receptor expression in mature neurons and responsiveness of these neurons to NTN and GDNF.

GF (TRN) receptors were also expressed in areas of WO 98/46622 PCT/US98/07996 71 the adult brain in which responsiveness to NTN and GDNF has not yet been directly evaluated. Areas of the brain in which Ret and one or both of the co-receptors were expressed include the oculomotor nucleus, the subthalamic nucleus, and several nuclei in the thalamus and hypothalamus. The strong correlation between receptor expression and responsiveness to NTN and GDNF suggests that neurons in these areas may respond to one or both factors.

Our finding that GF (TRN) receptors were expressed in areas of the hypothalamus involved in the control of feeding behavior and regulation of body weight, including the lateral hypothalamic area and the ventromedial and dorsomedial nuclei of the hypothalamus, suggests that NTN and/or GDNF may be involved in these processes. Previous studies that show weight loss in rats (Hudson et al., Brain Res. Bull. 35;425-432, 1995) and monkeys (Gash et al., J. Comp. Neurol. 363:345-358, 1995) treated with GDNF support such a role for GDNF. Other neurons that appear especially likely to benefit from the GF (TRN) factors are the motoneurons in the oculomotor nucleus; these neurons expressed all three GF (TRN) receptor components and their loss would be consistent with the ptosis observed in the NTN-deficient mice (Heuckeroth et al., Soc. Neurosci. Abstr. 23:668.1, 1997).

NTN and GDNF Expression Both NTN and GDNF were expressed in the targets of neurons that respond to these factors. As shown previously (Trupp et al., J. Neurosci. 17(10):3554-3567, 1997) and confirmed by the results reported here, GDNF mRNA is expressed in targets of dopaminergic neurons of the substantia nigra compacta (SNc); these targets include the olfactory tubercle, the nucleus accumbens, the striatum, and the globus pallidus. In addition, we found low-level expression of GDNF mRNA in other SN targets including the piriform cortex, and the arcuate, WO 98/46622 PCT/US98/07996 72 dorsomedial, and ventromedial nuclei of the hypothalamus.

We found GDNF mRNA in the compacta region of the substantia nigra, where GDNF protein may support dopaminergic neurons through an autocrine or paracrine mechanism. The present study also showed NTN mRNA expression in nigral targets including the arcuate and paraventricular nuclei of the hypothalamus. This distribution of NTN and GDNF mRNA is consistent with both of these growth factors functioning as target-derived trophic factors for nigral dopaminergic neurons in the mature brain.

NTN and GDNF were expressed diffusely in all layers of neocortex. The cerebral cortex may serve as a source of NTN and GDNF for a number of cortical-projecting neurons that express NTN and GDNF receptors; these include neurons in the nuclei of the medial septum and diagonal band of Broca that expressed Ret and GFRa-1 (TrnRl) and neurons in the locus coeruleus that express Ret and GFRa-1 (TrnRl) and GFRa-2 (TrnR2). NTN and GDNF mRNA were also expressed in a number of areas of the hippocampal formation including all three fields of Ammon's horn (CA1-3) within the hippocampus proper and in the dentate gyrus. In addition, GDNF mRNA was expressed in the subiculum. Like the neocortex, the hippocampus is a potential source of NTN and GDNF for neurons that project to this region of the brain and express GF receptors. Similarly, the medial septal nucleus and the nucleus of diagonal band of Broca, which also expressed GF (TRN) receptors, have large efferent projections to all fields of the hippocampal formation. The supramammillary nucleus of the hypothalamus in which all three GF (TRN) receptors are expressed could obtain NTN or GDNF by way of its efferents to the dentate gyrus and the hippocampus proper. The dentate gyrus and the hippocampus proper also receive a prominent projection from the locus coeruleus, which also expressed GF (TRN) WO 98/46622 PCT/US98/07996 73 receptors.

The strongest and most circumscribed NTN expression in the adult brain was seen in the lateral and medial magnocellular paraventricular nucleus and in the supraoptic nucleus of the hypothalamus. Within these nuclei are large magnocellular secretory neurons; these cells secrete oxytocin and vasopressin directly into the general circulation via the posterior pituitary. What function NTN may serve in these neurons is unclear.

Other growth factors, including fibroblast growth factor- 2 (Gonzales et al., Endocrinology 134(5):2289-2297, 1994) and insulin-like growth factor I (Aguado et al., Neuroendocrinol. 56:856-863, 1992) are found in hypothalamic magnocellular neurons and are believed to be involved in neuroendocrine function; NTN may serve a similar role. Alternatively, NTN in the supraoptic and paraventricular nuclei may be a source of target-derived trophic support for a number of neuronal populations that express GF (TRN) receptors and have efferent projections to these hypothalamic nuclei. For example, neurons in the medial preoptic nucleus and the medial and lateral septal nuclei project heavily to the supraoptic nucleus.

Cells in these nuclei expressed Ret mRNA as well as mRNA for GFRa-1 (TrnRl) and GFRa-2 (TrnR2). Afferent projections to the magnocellular neurons of the paraventricular nucleus are less well defined but are believed to include projections from the medial preoptic nucleus and the median and dorsal raphe nuclei. We found GF (TRN) receptor expression in each of these nuclei.

Another possibility is that NTN acts at some site distant to the hypothalamus and is secreted directly into the peripheral circulation like oxytocin and vasopressin.

Brain Regions That Do Not Contain Complete Receptor Complexes In our experiments as well as in previous studies (Nosrat et al., Exp. Brain Res. 115;410-422, 1997; Tomac et al., supra) of GF (TRN) receptor expression, WO 98/46622 PCT/US98/07996 74 incomplete receptor complexes have been found in some areas of the brain. For instance, in the thalamus and the trigeminal principal sensory nucleus, ret was expressed in the absence of GFRa-1 (TrnR) and GFRa-2 (TrnR2). In other regions, most strikingly neocortex and hippocampus, GFRa-1 (TrR1) or GFRa-2 (TrnR2) were expressed in the absence of Ret. These expression patterns raise the possibility that additional, as yet unidentified, receptor components for GDNF and NTN exist. Additional ligand-binding receptor components or other signaling components may exist in addition to Ret. This latter possibility seems less probable since all neurons that respond to NTN and GDNF express Ret.

An alternative possibility, which has been suggested previously (Trupp et al., supra), is that, in some cases, responsive neurons express Ret only and the ligand-binding component is supplied in trans perhaps by the target of the responsive neuron. Several patterns of NTN and GDNF receptor expression are consistent with this possibility. For example, in the principle thalamic motor (VL, VM) and sensory (VPM, VPL) relay nuclei, Ret was expressed in the absence of GFRa-1 (TrnR1) or GFRa-2 (TrnR2). Cells in these nuclei send projections to cerebral cortex where both GFRa-1 (TrR1) and GFRa-2 (TrnR2) were expressed at high levels. NTN and GDNF were also expressed in cerebral cortex. Therefore, thalamic neurons that express Ret may obtain NTN or GDNF as well as the ligand-binding receptor component from their target cortical neurons.

Another example of incomplete receptor expression was found in the Purkinje neurons of the cerebellum. In these neurons, only GFRa-2 (TrnR2) mRNA was expressed. Ret mRNA was expressed in other cells in the Purkinje layer, possibly basket cells or glial cells. The reason for this segregated expression in Purkinje neurons is unclear. Whether mature Purkinje neurons respond to NTN or GDNF is unknown, although embryonic Purkinje neurons respond to GDNF in vitro (Mount et al., Proc. Natl. Acad. Sci. 92:9092-9096, 1995).

Our findings show that GF (TRN) receptors are expressed in areas of the adult brain in which neurons that respond to these factors are located. In addition, GF (TRN) receptor expression in other areas suggests that additional NTN- or GDNF-responsive populations exist. In summary, the mRNA expression pattern WO 98/46622 PCT/US98/07996 for NTN, GDNF, and GF (TRN) receptors in the adult brain strongly suggests a role for these proteins as target-derived trophic factors for mature neurons.

In view of the above, it will be seen that the several advantages of the invention are achieved and other advantageous results attained.

As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

WO 98/46622 PCT/US98/07996 76 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: MILBRANDT, JEFFREY D JOHNSON JR, EUGENE M BALOH, ROBERT H (ii) TITLE OF INVENTION: TrnR2, A Novel Receptor Which Mediates Neurturin and GDNF Signaling Through Ret (iii) NUMBER OF SEQUENCES: 31 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: HOWELL HAFERKAMP, LC STREET: 7733 FORSYTH BLVD CITY: ST LOUIS STATE: MO COUNTRY: USA ZIP: 63105 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

-(viii) ATTORNEY/AGENT INFORMATION: NAME: HOLLAND, DONALD R REGISTRATION NUMBER: 35197 REFERENCE/DOCKET NUMBER: 976328 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 314-727-5188 TELEFAX: 314-727-6092 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1543 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 36..1427 (ix) FEATURE: NAME/KEY: sig_peptide LOCATION: 36..98 WO 98/46622 WO 9846622PCT/US98/07996 (xi) SEQUNCE DESCRIPTION: SEQ ID NO:1: GAGAAAGACA AAAAAAACGG TGGGATTTAT TTAAC ATG ATC TTG GCA AAC GTC Met Ile Leu Ala Asn Val -21

'I

TTC TGC CTC TTC TTC TTT CTA GAC GAG ACC Phe Cys Leu Phe Phe Phe Leu Asp Giu Thr -10 CTC CGC TCT TTG GCC Leu Arg Ser Leu Ala CCT TCC TCC Pro Ser Ser GAC TGT GTC Asp Cys Vai

CTG

Leu CAG GGC CCC GAG Gin Gly Pro Glu

CTC

Leu 10 CAC GGC TGG CGC His Gly Trp Arg CCC CCA GTG Pro Pro Val AAC TOC AGC Asn Cys Ser CGG GCC AAT GAG Arg Ala Asn Giu

CTG

Leu TGT GCC 0CC GAA Cys Ala Ala Giu TCT CGC Ser Arg TAC CGC ACT CTG Tyr Arg Thr Leu CAG TGC CTG GCA Gin Cys Leu Ala GGC CGC GAC CGC Giy Arg Asp Arg TTG GAG GTC TTG Leu Giu Val Leu

AAC

Asn

CAG

Gin

ACC

Thr so ATG CTG GCC AAC Met Leu Ala Asn GAG TGC CAG GCG Giu Cys Gin Ala

GCC

Ala 60 GAG AGC CCG CTG Glu Ser Pro Leu GAC TGC CGC TGC Asp Cys Arg Cys

AAG

Lys 75 CGG GGC ATG AAG Arg Gly Met Lys AAG GAG Lys Giu 149 197 245 293 341 389 437 485 533 581 CTG CAG TGT Leu Gin Cys GGT GAG GAG Gly Giu Glu 100 CAG ATC TAC TG Gin Ile Tyr Trp

AGC

Ser 90 ATC CAC CTG, GG Ile His Leu Gly TTC TAC GAA GCC Phe Tyr Giu Ala

TCC

Ser 105 CCC TAT GAG CCG Pro Tyr Glu Pro

GTG

Val 110 CTG ACC GAG Leu Thr Glu ACC TCC CGC Thr Ser Arg ACA GGG GCA Thr Giy Ala CTC TCG Leu Ser 115 GAC ATC TTC AGG ASP Ile Phe Arg GCT TCA ATC TTC Ala Ser Ile Phe 4, TCA GG Ser Gly 125

GAC

Asp 130 CCG GTG GTC AGC Pro Val Val Ser GCC AAG Ala Lys 135 GAC AAC Asp Asn AGC AAC CAT Ser Asn His TGC AAG AAG Cys Lys Lys 155

TGC

Cys 140 CTG GAT GCT GCC Leu Asp Ala Ala GCC TGC AAC CTG Ala Cys Asn Leu CTG CGC TCC TCC Leu Arg Ser Ser TAC ATC Tyr Ile 160 TCC ATC TGC Ser Ile Cys AAG TGC CAC Lys Cys His 180

AAC

Aen 165 CGC GAG ATC TCG Arg Glu Ile Ser ACC GAG CGC TGC Thr Giu Arg Cys AAC CGC CGC Asn Arg Arg 175 CCC AGC GAG Pro Ser Glu 629 677 AAG GCC CTG CGC Lys Ala Leu Arg

CAG

Gin 185 TTC TTC GAC CG Phe Phe Asp Arg WO 98/46622 WO 9846622PCTIUS98/07996 TAC ACC Tyr Thr 195 TAC CGC ATG CTC Tyr Arg Met Leu

TTC

Phe 200 TGC TCC TGC CAA Cys Ser Cys Gin CAG GCG TGC GCT Gin Ala Cys Ala

GAG

Glu 210

GAG

Giu CGC CGC CGG CAA Arg Arg Arg Gin AAG CCC AAC TGC Lys Pro Asn Cys 230

ACC

Thr 215 ATC CTG CCC AGC Ile Leu Pro Ser TCC TAT GAG GAC Ser Tyr Glu Asp

AAG

Lys 225 CTG GAC CTG CGT Leu Asp Leu Arg GTG TGC CGG ACT Val Cys Arg Thr GAC CAC Asp His 240 CTG TGT CGG Leu Cys Arg TAC CAG ACG Tyr Gin Thr 260

TCC

Ser 245 CGG CTG GCC GAC Arg Leu Ala Asp CAT GCC AAT TGT His Ala Asn Cys CGA GCC TCC Arg Ala Ser 255 GCG TGT CTG Ala Cys Leu GTC ACC AGC TGC Val Thr Ser Cys

CCT

Pro 265 GCG GAC AAT TAC Ala Asp Asn Tyr GGC TCT Gly Ser 275 TAT GCT GGC ATG Tyr Ala Gly Met

ATT

Ile 280 GGG TTT GAC ATG Gly Phe Asp Met CCT AAC TAT GTG Pro Asn Tyr Val

GAC

Asp 290 TCC AGC CCC ACT Ser Ser Pro Thr

GGC

Gly 295 ATC GTG GTG TCC Ile Val Val Ser TGG TGC AGC TGT Trp Cys Ser Cys

COT

Arg 305 GGC AGC GGG AAC Gly Ser Gly Asn GAG GAG GAG TGT Glu Giu Giu Cys GAG AAG Glu Lys 315 TTC CTC AGO GAC TTC Phe Leu Arg Asp Phe 320 ACC GAG AAC Thr Glu Asn ACG GAC GTG Thr Asp Val 340 CCA TGC CTC COG AAC GCC ATC CAG GCC TTT GGC AAC GGC Pro Cys Leu Arg Asn Ala Ile Gin Ala Phe Oly Asn Gly 325 330 335 AAC GTG TCC CCA Asn Val Ser Pro

AAA

Lys 345 GOC CCC TCG TTC Gly Pro Ser Phe

CAG

Gin 350 0CC ACC CAG Ala Thr Gin 869 917 965 1013 1061 1109 1157 1205 1253 1301 1349 1397 1447 1507 GCC CCT COG OTO GAG AAG, ACO CCT TCT TTG CCA GAT GAC CTC AGT GAC Ala Pro Arg Val Giu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser Asp 355 360 365

AGT

Ser 370 ACC AGC TTG 000 Thr Ser Leu Gly

ACC

Thr 375 AGT GTC ATC ACC Ser Val Ile Thr TOC ACG TCT GTC Cys Thr Ser Val

CAG

Gin 385 GAG CAG 000 CTG Giu Gin Gly Leu 0CC AAC AAC TCC Ala Asn Asn Ser

AAA

Lys 395 GAG TTA AGC ATG Glu Leu Ser Met TGC TTC Cys Phe 400 ACA GAG Thr Giu CCT AAC Pro Asn CTG TCT Leu Ser 435

CTC

Leu ACA AAT ATC ATC Thr Asn Ile Ile

CCA

Pro 410 000 AGT AAC Oly Ser Asn AAG OTO ATC AAA Lys Val Ile Lys 415 0CC TTG ACC GTO Ala Leu Thr Val 430 TCA GGC CCC AGC Ser Giy Pro Ser 420 GTC CTO ATO CTG Val Leu Met Leu

AGA

Arg 0CC Ala 425 AGA CCG TCG OCT Arg Pro Ser Ala AAA CAG GCC TT6'TAGGCTGTGG GAACCGAGTC Lys Gin Ala Leu 440 AGAAGATTTT TGA1AAGCTAC GCAGACAAGA ACAGCCGCCT GACGAAATGG AAACACACAC WO 98/46622 PCTIUS98/07996 79 AGACACACAC ACACCTTGCA AAAAAA AAAAAA 1543 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 464 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ile Leu Ala Asn Val Phe Cys Leu Phe Phe Phe Leu Asp Glu Thr -21 -20 -15 Leu Arg Ser Leu Ala Ser Pro Ser Ser Leu Gin Gly Pro Glu Leu His 1 5 Gly Trp Arg Pro Pro Val Asp Cys Val Arg Ala Asn Giu Leu Cys Ala 20 Ala Giu Ser Asn Cys Ser Ser Arg Tyr Arg Thr Leu Arg Gin Cys Leu 35 Ala Gly Arg Asp Arg Asn Thr Met Leu Ala Asn Lys Giu Cys Gin Ala 50 Ala Leu Giu Val Leu Gin Giu Ser Pro Leu Tyr Asp Cys Arg Cys Lys 65 70 Arg Gly Met Lys Lys Giu Leu Gin Cys Leu Gin Ile Tyr Trp Ser Ile 85 His Leu Gly Leu Thr Glu Gly Glu Giu Phe Tyr Giu Ala Ser Pro Tyr 100 105 Glu Pro Val Thr Ser Arg Leu Ser Asp Ile Phe Arg Leu Ala Ser Ile 110 115 120 Phe Ser Gly Thr Gly Ala Asp Pro Val Val Ser Ala Lys Ser Asn His 125 130 135 Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys 140 145 150 155 Leu Arg Ser Ser Tyr Ile Ser Ile Cys Asn Arg Giu Ile Ser Pro Thr 160 165 170 Glu Arg Cys Aen. Arg Arg Lys Cys His Lys Ala Leu Arg Gin Phe Phe 175 180 185 Asp Arg Val Pro Ser Giu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys 190 195 200 Gin Asp Gin Ala Cys Ala Giu Arg Arg Azrg Gin Thr Ile Leu Pro Ser 205 210 215 Cys Ser Tyr Giu Asp Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Gly 220 225 230 235 Val Cys Arg Thr Asp His Leu Cys Arg Ser Arg Leu Ala Asp Phe His 240 245 250 WO 98/46622 WO 9846622PCT/US98/07996 Ala Asn Cys Arg Ala Ser Tyr Gln Thr Val Thr Ser Cys Pro Ala Asp 255 260 265 Asn Tyr Gin Ala Cys Leu Gly Ser Tyr Ala Gly Met Ile Giy Phe Asp 270 275 280 Met Thr Pro Asn Tyr Val Asp Ser Ser Pro Thr Gly Ile Val Val Ser 285 290 295 Pro Trp Cys Ser Cys Arg Gly Ser Gly Asn Met Glu Glu Giu Cys Glu 300 305 310 31S Lys Phe Leu Arg Asp Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala Ile 320 325 330 Gin Ala Phe Gly Asn Gly Thr Asp Val Asn Val Ser Pro Lys Gly Pro 335 340 345 Ser Phe Gin Ala Thr Gin Ala Pro Arg Val Giu Lys Thr Pro Ser Leu 350 355 360 Pro Asp Asp Leu Ser Asp Ser Thr Ser Leu Gly Thr Ser Val Ile Thr 365 370 375 Thr Cys Thr Ser Val Gin Giu Gin Giy Leu Lye Ala Asn Asn Ser Lye 380 385 390 395 Giu Leu Ser Met Cys Phe Thr Glu Leu Thr Thr Asn Ile Ile Pro Gly 400 405 410' Ser Asn Lys Val Ile Lye Pro Asn Ser Gly Pro Ser Arg Ala Arg Pro 415 420 425 Ser Ala Ala Leu Thr Val Leu Ser Val Leu Met Leu Lye Gin Ala Leu 430 435 440 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 411 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Ser Pro Ser Ser Leu Gin Gly Pro Glu Leu His Gly Trp Arg Pro Pro 1 5 10 Val Asp Cys Val Arg Ala Asn Giu Leu Cys Ala Ala Glu Ser Aen Cys 25 Ser Ser Arg Tyr Arg Thr Leu Arg Gin Cys Leu Ala Gly Arg Asp Arg 40 Asn Thr Met Leu Ala Asn Lys Glu Cys Gin Ala Ala Leu Glu Val Leu 55 WO 98/46622 WO 9846622PCTIUS98/07996 Gin Giu Ser-Pro Leu Tyr 70 Asp Cys Arg Cys Lys Arg Gly Met Lys Lys Giu Leu Giu Gly Arg Leu Ala Asp 130 Lys Ala 145 Ile Ser Arg Lys Giu Tyr Gin Glu Ser 115 Pro Cys Ile Cys Thr 195 Cys Giu 100 Asp Val Asn Cys His 180 Tyr Leu Phe Ile Val1 Leu Asn 165 Lys Arg Gin Tyr Phe Ser Asn 150 Arg Ala Met Ile Glu Arg Ala 135 Asp Glu Leu Leu Tyr Ala Leu 120 Lys Asn Ile Arg Phe 200 Trp Ser 105 Ala Ser Cys Ser Gin 185 Cys Ser 90 Pro Ser Asn Lys Pro 170 Phe Ser Pro Ile His Leu Gly Leu Thr Tyr Giu Ile Phe His Cys 140 Lys Leu 155 Thr Glu Phe Asp Cys Gin Ser Cys 220 Gly Val 235 His Ala Pro Ser 125 Leu Arg Arg Arg Asp 205 Ser Cys Asn Val 110 Gly Asp Ser Cys Val 190 Gin Tyr Arg Cys Thr Thr Ala Ser Asn 175 Pro Ala Glu Thr Arg 255 Ser Giy Ala Tyr 160 Arg Ser Cys Asp Asp 240 Ala Cys Ala Giu Arg Arg Arg 210 Gin Thr Ile Leu 215 Lys 225 Glu Lys Pro Asn Cys 230 Leu Asp Leu Arg His Leu Cys Arg Ser Arg Leu Ala Asp Phe 245 250 Ser Tyr Gin Thr Val Thr Ser Cys Pro Ala Asp Asn Tyr Gin Ala 260 265 Leu Gly Ser Tyr Ala Gly Met Ile Gly 275 280 Val Asp Ser Ser Pro Thr Gly Ile Val 290 295 Arg Gly Ser Gly Asn Met Giu Giu Glu 305 310 Phe Thr Giu Asn Pro Cys Leu Arg Asn 325 Gly Thr Asp Val Asn Val Ser Pro Lys 340 345 Gin Ala Pro Arg Val Giu Lys Thr Pro 355 360 Asp Ser Thr Ser Leu Gly Thr Ser Val 370 375 Gin Glu Gin Gly Leu Lys Ala Asn Asn 385 390 Phe Thr Giu Leu Thr Thr Asn Ile Ile 270 Phe V1al Cys Ala 330 Gly Ser Ile Ser Prc 4102 Asp Ser Giu 315 Ile Pro Leu Thr Lys 395 Gij Met Thr 285 Pro Trp 300 Lys Phe Gin Ala Ser Phe Pro Asp 365 Thr Cys 380 Giu Leu Pro Cys Leu Phe Gin 350 Asp Thr Ser Asn Ser Arg Gly 335 Ala Leu Ser Met Tyr Cys Asp 320 Asn Thr Ser Val Cys 400 405 WO 98/46622 WO 9846622PCT/US98/07996 82 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 1392 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:

NAME/KEY:.

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

CDS

1. .1389 sigjpeptide 1. .63 matpeptide 64. .1389 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ATG ATC Met Ile -21 -20 TTG GCA AAC CC Leu Ala Asn Ala TTC TGC Phe Cys -i5 CTC TTC TTC TTT TTA GAC GAA ACC 48 Leu Phe Phe Phe Leu Asp Glu Thr CTC CGC TCT TTG CCC ACC CCT TCC TCT CCG CAG CCC TCT GAG CTC CAC Leu Arg Ser Leu Ala Ser Pro Ser Ser Pro Gin Gly Ser Giu Leu His 1 5 GGC TGG CGC Gly Trp, Arg GCT GAA TCC Ala Glu Ser CAA GTG GAC TCT Gin Val Asp Cys GTC CCC Val Arg CCC AAT GAG Ala Asn Giu CTC TGT CC Leu Cys Ala CAG TGC CTG Gin Cys Leu AAC TGC AGC TCC Asn Cys Ser Ser TAC.CCC ACC CTT Tyr Arg Thr Leu GCC GCC Ala Gly CCC CAT CCC AAT Arg Asp Arg Asn ATG CTC GCC R.AT Met Leu Ala Asn GAG TGC CAG CC Giu Cys Gin Ala

CC

Ala CTG GAG GTC TTG Leu Glu Val Leu GRA AGC CCA TTG Giu Ser Pro Leu GAC TGC CCC TGC Asp Cys Arg Cys

RAG

Lys CCC CCC ATO RAG, Arg Gly Met Lys GAG CTG CAG TGT Glu Leu Gin Cys

CTG

Leu CAG ATC TAT TCC Gin Ile Tyr Trp AGC ATC Ser Ile 240 288 336 384 432 CAT CTG GGG CTG ACG GAG GGT GAG GAG TTC TAC GAA OCT TCG CCC TAT His Leu Gly Leu Thr Glu Gly Clu Clu Phe Tyr Glu Ala Ser Pro Tyr 100 105 GAG CCT CTG ACC TCC CCC CTC TCG GAC ATC TTC AGG CTC GCT TCA ATC Glu Pro Val Thr Ser Arg Leu Ser Asp Ile Phe Arg Leu Ala Ser Ile 110 115 120 WO 98/46622 WO 9846622PCT[US98/07996 TTC TCA Phe Ser 125 GGG ACA GGG GCA Gly Thr Gly Ala

GAC

Asp 130 CCG GTG GTC AGT Pro Val Val Ser GCC AAG AGC AAC CAC Ala Lys Ser Asn His 135 GAC AAC TGC AAG, AAG Asp Asn Cys Lys Lys

TGC

Cys 140 CTG GAT GCC GCC Leu Asp Ala Ala GCC TGC AAC CTG Ala Cys Asn Leu CTC CGC TCC TCC Leu Arg Ser Ser

TAC

Tyr 160 ATC TCC ATC TGC Ile Ser Ile Cys CGC GAG ATC TCT Arg Giu Ile Ser CCC ACT Pro Thr 170 GAA CGC TGC Glu Arg Cys GAC CGT GTG Asp Arg Val 190

AAC

Asn 175 CGC CGC AAG TGC Arg Arg Lys Cys

CAC

His 180 AAG GCC CTG CGC Lys Ala Leu Arg CAG TTC TTC Gin Phe Phe 185 TGC TCC TGT Cys Ser Cys CCC AGC GAG TAT Pro Ser Giu Tyr

ACC

Thr 195 TAC CGC ATG CTC Tyr Arg Met Leu

TTC

Phe 200 CAG GAC Gin Asp 205 CAG GCA TGC GCC Gin Ala Cys Ala

GAG

Giu 210 CGT CGC CGG CAA Arg Arg Arg Gin

ACC

Thr 215 ATC CTG CCC AGC Ile Leu Pro Ser

TGT

Cys 220 TCC TAT GAG GAC Ser Tyr Giu Asp

AAG

Lys 225 GAG AAG CCC AAC Glu Lys Pro Asn TTG GAC CTG CGC Leu Asp Leu Arg

AGC

Ser 235 CTG TGT CGT ACA Leu Cys Arg Thr

GAC

Asp 240 CAC TTG TGC CGG His Leu Cys Arg CGC CTG GCA GAG Arg iLeu Ala Asp TTC CAC Phe His 250 480 528 576 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 GCC AAC TGT Ala Asn Cys AAC TAC CAG Asn Tyr Gin 270

CGA

Arg 255 GCC TCC TAG CGG Ala Ser Tyr Arg

ACA

Thr 260 ATC ACC AGC TGC Ile Thr Ser Cys GCA TGT CTG GGC Ala Cys Leu Giy TAT GCT GGC ATG Tyr Ala Gly Met CCT GCG GAG Pro Ala Asp 265 GGG TTT GAT Giy Phe Asp GTG GTG TCT Val Val Ser ATG ACA Met Thr 285 CCG AAC TAT GTG Pro Asn Tyr Val TCC AAC CCC ACG Ser Asn Pro Thr GGC ATC Gly Ile 295

CCC

Pro 300 TGG TGC AAT TGT Trp Cys Asn Gys GOC AGT GGG AAC Gly Ser Gly Asn

ATG

Met 310 GAA GAA GAG TGT Giu Glu Giu Cys

GAG

Glu 315 AAG TTC CTC AAG Lys Phe Leu Lys TTC ACA GAA AAC Phe Thr Glu Asn TGC CTC CGG AAT Cys Leu Arg Asn GCC ATT Ala Ile 330 CAA GCC TTT Gin Ala Phe ACA TTT TCA Thr Phe Ser 350

GGC

Gly 335 AAT GGC ACA GAT Asn Gly Thr Asp AAC ATG TCT CCC Asn Met Ser Pro AAA GGC CCC Lys Gly Pro 345 CCT TCA CTG Pro Ser Leu GCT ACC GAG GCC Ala Thr Gln Ala CGG GTA GAG AAA Arg Val Glu Lys

ACT

Thr 360 CGA GAT Pro Asp 365 GAG CTG AGT GAT Asp Leu Scr Asp AGG ACC AGC CTG GGG ACC Ser Thr Scr Leu Gly Thr 370 375 AGT GTC ATG ACC Ser Val Ile Thr WO 98/46622 WO 9846622PCT/US98/07996

ACC

Thr 380

GAG

Giu

AGT

Ser

TCG

Ser

TAG

TGC ACA TCT ATC CAG GAG CAA GGG CTG AAG GCC AAC AAC TCC AAA Cys Thr Ser Ile Gin Giu Gin Giy Leu Lys Ala Asn Asn Ser Lys 385 390 395 TTA AGC ATG TGT TTC ACA GAG CTC ACG ACA AAT ATC AGC CCA GGG Leu Ser Met Cys Phe Thr Giu Leu Thr Thr Asn Ile Ser Pro Gly 400 405 410 AAA AAG GTG ATC AAA CTT TAC TCA GGC TCC TGC AGA GCC AGA CTG Lys Lys Val Ile Lys Leu Tyr Ser Gly Ser Cys Arg Ala Arg Leu 415 420 425 ACT GCC TTG ACT GCC CTC CCA CTC CTG ATG GTG ACC TTG GCC Thr Ala Leu Thr Ala Leu Pro Leu Leu Met Val Thr Leu Ala 430 435 440 1248 1296 1344 1389 1392 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 463 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N Met Ile Leu Ala Asn Ala Phe Cys Leu Phe P -21 -20 -15 Leu Arg Ser Leu Ala Ser Pro Ser Ser Pro G 1 5 Gly Trp Arg Pro Gin Val Asp Cys Val ArgX 20 Ala Glu Ser Asn Cys Ser Ser Arg Tyr Arg J 35 Ala Gly Arg Asp Arg Asn Thr Met Leu Ala so Ala Leu Glu Val Leu Gin Glu Ser Pro Leu 65 Arg Gly Met Lys Lys Glu Leu Gin Cys Leu 85 His Leu Gly Leu Thr Giu Gly Glu Glu Phe 100 Glu Pro Val Thr Ser Krg Leu Ser Asp Ile 110 115 Phe Ser Gly Thr Gly Ala Asp Pro Val Val 125 130 Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu 140 145 Leu Arg Ser Ser Tyr Ile Ser Ile Cys Asn 160 165 'he Phe Leu A ~In Gly Ser G ~la Asn Giu I Ehr Leu ArgC ksn Lys Giu Tyr Asp Cys 70 Gin Ile Tyr Tyr Glu Ala Phe Arg Leu 120 Ser Ala Lys 135 Asn Asp Asn 150 Arg Glu Ile sp Glu Thr 1 1u Leu His ~eu Cys Ala 1n Cys Leu ys Gin Ala Arg Cys Lys Trp Ser Ile Ser Pro Tyr 105 Ala Ser Ile Ser Asn His Cys Lys Lys 155 Ser Pro Thr 170 WO 98/46622 WO 9846622PCTIUS98/07996 Giu Asp Gin Cys 220 Leu Ala Asn Met Pro 300 Lys Gln Thi Pr Th: 384 Gli Se Se (2 Arg Cys Asn Arg Arg Lys Cys His Lys Ala 175 180 Arg Vai pro Ser Glu Tyr Thr Tyr Arg Met 190 195 Asp Gin Ala Cys Ala Giu Arg Arg Arg Gir 205 210 Ser Tyr Giu Asp Lys Giu Lys Pro Asn CyE 225 23( Cys Arg Thr Asp His Leu Cys Arg Ser Arc 240 245 Asn Cys Arg Ala Ser Tyr Arg Thr Ile Th: 255 260 Tyr Gin Ala Cys Leu Gly Ser Tyr Ala Gi' 270 275 Thr Pro Asn Tyr Val Asp Ser Asn Pro Th 285 290 Trp Cys Asn Cys Arg Gly Ser Gly Asn Me 305 31 Phe Leu Lys Asp Phe Thr Giu Asn Pro Cy 320 325 Ala Phe Gly Asn Gly Thr Asp Val Asn Me 335 340 Phe Ser Ala Thr Gin Ala Pro Arg Val G] 350 355 Asp Asp Leu Ser Asp Ser Thr Ser Leu G 365 370 r Cys Thr Ser Ile Gin Glu Gin Gly Leu L' 0 3853 Leu Ser Met Cys Phe Thr Glu Leu Thr T 400 405 r Lys Lys Val Ile Lys Leu Tyr Ser Gly S 415 420 Thr Ala Leu Thr Ala Leu Pro Leu Leu M 430 435 .INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 411 amino acids TYPE: amino acid STRAlNDEDNESS: not relevant TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES Leu Arg Gin P1 185 Leu Phe Cys Si 200 Thr Ile Leu P 215 Leu Asp Leu A 3 Leu Ala Asp P 2 r Ser Cys Pro A 265 y Met Ile Gly P 280 r Gly Ile Val 295 t Glu Giu GluC 0 s Leu Arg Asn ~t Ser Pro Lys 345 -u Lys Thr Pro 360 Ly Thr Ser Val 375 ys Ala Asn Asn 90 hr Asn Ile Ser er Cys Arg Ala 425 Let Val Thr Leu 440 hie ro rg he la ihe ~al :ys kla 330 Gly Ser Ile Se Pr 414 Ax Al Phe Cys Ser Ser 235 His Asp Asp Ser Giu 315 Ile *Pro *Leu Thr *Lys 395 )Gly 0 g Leu a WO 98/46622 WO 9846622PCTIUS98/07996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Ser Pro Ser Ser Pro Gin Gly Ser Giu Leu His Gly Trp Arg Pro Gin 1 5 10 Val1 Ser Asn Gin Glu Glu Arg Ala Asp Ser Thr Giu Leu Gly Leu Asp 130 Cys Arg Met Ser Gin Giu Ser 115 Pro Val Tyr Leu Pro Cys Giu 100 Asp Val Arg Ala Leu Leu Phe Ile Val Ala Thr Asn Tyr 70 Gin Tyr Phe Ser Asn Leu Ly s 55 Asp Ile Giu Arg Ala 135 Giu Arg 40 Giu Cys Tyr Ala Leu 120 Lys Leu 25 Gin Cys Arg Trp Ser 105 Ala Ser Cys Cys Gin Cys Ser 90 Pro Ser Asn Ala Leu Ala Lys 75 Ile Tyr Ile His Ala Ala Ala Arg His Giu Phe Cys 140 Giu Gly Leu Gly Leu Pro Ser 125 Leu Ser Arg Giu Met Gly Val 110 Gly Asp Asn Asp Val Lys Leu Thr Thr Ala Cys Arg Leu Lys Thr Ser Gly Ala Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser Tyr 150 155 160 Ile S ArgI Giu Ala Lys 225 His Ser Leu Val Arg 305 Phe er IleC ,ys Cys I'yr Thr 195 G1u Arg 210 Giu Lys Leu Cys Tyr Arg Gly Ser 275 Asp Ser 290 Gly Ser Thr Giu ~ys Us ryr Arg Pro Arg Thr 260 Ty2 Asi G1' As: zgsn 7 165 Lys2 Arg Arg Asn Ser 245 Ile Ala i Pro y Asn n Pro 32S ~rg 1.ia ,4et Gln Cys 230 Arg Thr Gly Thr Met 310 Cy~ Giu Leu Leu Thr 215 Leu Leu Ser Met G1~ 29! Gli 3 Le Ile Arg Phe 200 Ile Asp Ala Cys Ile 280 u Gl.

u Arc Ser Pro Thr 170 Gin Phe Phe 185 Cys Ser Cys Leu Pro Ser Leu Arg Ser 235 Asp Phe His 250 Pro Ala Asp 265 Gly Phe Asr Val Val Sei Giu Cys Gli 3i1! g Asn Aia Ill 330 Glu Arg C Asp Arg N Gin Asp 205 Cys Ser 220 Leu Cys Ala Asn Asn Tyr Met Thr 285 Pro Trp 300 i Lys Phe a Gin Ala :ys lal .,m Tyr Arg Cys Gin 270 Pro CyE Let Ph Asn 175 Pro Ala Glu.

Thr Arg 255 Ala Asn Asn i Lys e Gly 335 1 .rg 3er Dys Asp Asp 240 Ala Cys Tyr Cys Asp 320 As n WO 98/46622 PCT/US98/07996 87 Gly Thr Asp Val Asn Met Ser Pro Lys Gly Pro Thr Phe Ser Ala Thr 340 345 350 Gin Ala Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser 355 360 365 Asp Ser Thr Ser Leu Gly Thr Ser Val Ile Thr Thr Cys Thr Ser Ile 370 375 380 Gin Glu Gin Gly Leu Lys Ala Asn Asn Ser Lys Glu Leu Ser Met Cys 385 390 395 400 Phe Thr Glu Leu Thr Thr Asn Ile Ser Pro Gly 405 410 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 331 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Met Ile Leu Ala Asn Val Phe Cys Leu Phe Phe Phe Leu Gly Thr Gly 1 5 10 Ala Asp Pro Val Val Ser Ala Lys Ser Asn His Cys Leu Asp Ala Ala 25 Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser Tyr 40 Ile Ser Ile Cys Asn Arg Glu Ile Ser Pro Thr Glu Arg Cys Asn Arg 55 Arg Lys Cys His Lys Ala Leu Arg Gin Phe Phe Asp Arg Val Pro Ser 70 75 Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gin Asp Gin Ala Cys 90 Ala Glu Arg Arg Arg Gin Thr Ile Leu Pro Ser Cys Ser Tyr Glu Asp 100 105 110 Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Gly Val Cys Arg Thr Asp 115 120 125 His Leu Cys Arg Ser Arg Leu Ala Asp Phe His Ala Asn Cys Arg Ala 130 135 140 Ser Tyr Gin Thr Val Thr Ser Cys Pro Ala Asp Asn Tyr Gin Ala Cys 145 150 155 160 Leu Gly Ser Tyr Ala Gly Met Ile Gly Phe Asp Met Thr Pro Asn Tyr 165 170 175 WO 98/46622 PCT/US98/07996 88 Val Asp Ser Ser Pro Thr Gly Ilie Val Val Ser Pro Trp Cys Ser Cys 180 185 190 Arg Gly Ser Gly Asn Met Giu Giu Giu Cys Giu Lys Phe Leu Arg Asp 195 200 205 Phe Thr Giu Asn Pro Cys Leu Arg Asn Ala Ile Gin Ala Phe Gly Asn 210 215 220 Gly Thr Asp Val Asn Val Ser Pro Lys Gly Pro Ser Phe Gin Ala Thr 225 230 235 240 Gin Ala Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser 245 250 255 Asp Ser Thr Ser Leu Gly Thr Ser Val Ile Thr Thr Cys Thr Ser Val 260 265 270 Gin Giu Gin Gly Leu Lys Ala Asn Asn Ser Lys Giu Leu Ser Met Cys 275 280 285 Phe Thr Glu Leu Thr Thr Asn Ilie Ile Pro Gly Ser Asn Lys Val Ile 290 295 300 Lys Pro Asn Ser Gly Pro Ser Arg Ala Arg Pro Ser Ala Ala Leu Thr 305 310 315 320 Val Leu Ser Val Leu Met Leu Lys Gin Ala Leu 325 330 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 330 amino acids TYPE: amino acid STRA1NDEDNESS: not relevant TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Ile Leu Ala Asn Ala Phe Cys Leu Phe Phe Phe Leu Gly Thr Giy 1 S 10 Ala Asp Pro Val Val Ser Ala Lys Ser Asn His Cys Leu Asp Ala Ala 25 Lys Ala Cys Asn Leu Asn Asp Aen Cys Lys Lys Leu Arg Ser Ser Tyr 40 Ile Ser Ile Cys Asn Arg Giu Ile Ser Pro Thr Giu Arg Cys Asn Arg 55 Arg Lys Cys His Lys Ala Leu Arg Gin Phe Phe Asp Arg Val Pro Ser 70 75 Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gin Asp Gin Ala Cys 90 Ala Giu Arg Arg Arg Gin Thr Ile Leu Pro Ser Cys Ser Tyr Glu Asp 100 105 110 WO 98/46622 PCT/US98/07996 89 Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Ser Leu Cys Arg Thr Asp 115 120 125 His Leu Cys Arg Ser Arg Leu Ala Asp Phe His Ala Asn Cys Arg Ala 130 135 140 Ser Tyr Arg Thr Ile Thr Ser Cys Pro Ala Asp Asn Tyr Gin Ala Cys 145 150 155 160 Leu Gly Ser Tyr Ala Gly Met Ile Gly Phe Asp Met Thr Pro Asn Tyr 165 170 175 Val Asp Ser Asn Pro Thr Gly Ile Val Val Ser Pro Trp Cys Asn Cys 180 185 190 Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu Lys Phe Leu Lys Asp 195 200 205 Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala Ile Gin Ala Phe Gly Asn 210 215 220 Gly Thr Asp Val Asn Met Ser Pro Lys Gly Pro Thr Phe Ser Ala Thr 225 230 235 240 Gin Ala Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser 245 250 255 Asp Ser Thr Ser Leu Gly Thr Ser Val Ile Thr Thr Cys Thr Ser Ile 260 265 270 Gin Glu Gin Gly Leu Lys Ala Asn Asn Ser Lys Glu Leu Ser Met Cys 275 280 285 Phe Thr Glu Leu Thr Thr Asn Ile Ser Pro Gly Ser Lys Lys Val Ile 290 295 300 Lys Leu Tyr Ser Gly Ser Cys Arg Ala Arg Leu Ser Thr Ala Leu Thr 305 310 315 320 Ala Leu Pro Leu Leu Met Val Thr Leu Ala 325 330 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Cys Arg Cys Lys Arg Gly Met Lys Lys Glu 1 5 WO 98/46622 PCT/US98/07996 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID Cys Asn Arg Arg Lys Cys His Lys Ala Lys Arg 1 5 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO FRAGMENT TYPE: internal (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 3 OTHER INFORMATION: /product= "OTHER" /note= "Xaa is Lys or Arg" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l1: Cys Leu Xaa Asn Ala Ile Glu Ala Phe Gly Asn Gly 1 5 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 465 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu Leu 1 5 10 WO 98/46622 PCTIUS98/07996 Leu Se Ser As Leu Az Gly LE Gin L~ Lys A Asn A Ser A 1 Vai G 145 Cys A Pro C His I Tyr Arg 225 Pro Arg Ser Tyr Ser 305 Asp Cys Val p

(S

uu (s sn sp sp 30 lu .sn Se

VE

1.71 21( Are Asl Se Va Se 2S

T(

Lt

T

Ala Glu Va Gin Cys Le Gin Cys Va Glu Ala Ly Ser Leu T) Cys Leu Al 100 Leu Leu G1 115 Ile Phe A His Ile P Leu Asp A 1 Thr Thr S 180 Ala Leu A 195 r Met Leu I 0 g Gin Thr n Cys Leu r Arg Leu 260 .1 Ser Ser 275 .r Gly Leu 10 r Leu Ser 4u Glu Glu eu Lys Asn 340 rp Gin Pro 355 1 u .1 -5 rr :g Lu rg ro sp 65 er rTg )he Se~ 24 Al; Cy Ii Va 3:

A:

A

Ser Gly Gly Asp Arg Leu As 25 Lys Giu Gin Ser Cys Ser Th 40 Ala Gly Lys Giu Thr Asn P1 55 Asp Glu Cys Arg Ser Ala ME 70 75 Asn Cys Arg Cys Lys Arg G Ile Tyr Trp Ser Met Tyr G 105 Asp Ser Pro Tyr Giu Pro V 120 Val Val Pro Phe Ile Ser A 135 1 Ly s Gly Asn Asn Cys Leu A 150 155 Ile Cys Lys Lys Tyr Arg S 170 Val Ser Asn Asp Val Cys A 185 Gin Phe Phe Asp Lys Val P 200 Cys Ser Cys Arg Asp Ile 7 215 4 3 Val Pro Val Cys Ser Tyr 230 235 r Leu Gin Asp Ser Cys Lys 5 250 a Asp Phe Phe Thr Asn Cys 265 s Leu Lys Giu Asn Tyr Ala 280 e Gly Thr Val Met Thr Pro 295 1i Ala Pro Trp Cys Asp Cys 310 315 r8 Leu Lys Phe Leu Asn Phe as 330 La Ile Gin Ala Phe Gly Asn 345 la Pro Pro Val Gin Thr Thr 360 p Cys ir Lys Le Ser it Glu Ly Met Ln Ser al Asn 125 sp Val sp Ala er Ala ,sn Arg 'ro Ala 205 la Cys 31u GL.

rhr Asi Gin Pr Asp Cy 28 Asn Ty 300 Ser As Phe L Gly SE Thr A: 31 Val Lys Ala Tyr Arg Thr Leu Ala Ser Ala Leu Lys Lys Lys Glu Leu Gin Gly 110 Ser Arg Leu Phe Gin Gin Ala -Lys Ala 160 Tyr Ile Thr 175 Arg Lys Cys 190 Lys His Ser Thr Giu Arg 1 Arg Glu Lys 240 i Tyr Ile Cys 255 D Glu Ser Arg 270 s Leu Leu Ala r Ile Asp Ser n Ser Gly Asn 320 rs Asp Asn Thr 335 r Asp Val Thr 350 La Thr Thr Thr WO 98/46622 WO 9846622PCT/US98/07996 Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gl 370 375 Asn Giu Ile Pro Thr His Val Leu Pro Pro Cy 38S 390 39 Gin Lys Leu Lys Ser Asn Val Ser Gly Asn Th 405. 410 Asn Gly Asn Tyr Glu Lys Giu Gly Leu Gly Al 420 425 Thr Lys Ser Met Ala Ala Pro Pro Ser Cys GI 435 440 Val Leu Val Val Thr Ala Leu Ser Thr Leu Le 450 45S Ser 465 INFORMATION FOR SEQ ID NO:l3: SEQUENCE CHARACTERISTICS: LENGTH: 468 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu I 1 5 10 Met Ser Ala Glu Val Ser Gly Gly Asp Arg 25 Ser Asp Gin Cys Leu Lys Glu Gin Ser Cys 40 Leu Arg Gin Cys Val Ala Gly Lys Glu Thr 5 Gly Leu Giu Ala Lys Asp Glu Cys Arg Ser 70 Gin Lys Ser Leu Tyr Asn Cys Arg Cys Lys 90 Lys Asn Cys Leu Arg Ile Tyr Trp Ser Met 100 105 Asn Asp Leu Leu Giu Asp Ser Pro Tyr Glu 115 120 Ser Asp Ilie Phe Arg Ala Vai Pro Phe Ile 130 135 y r a Pro Ala 380 Ala Asn His Leu Ser Ser Leu Ser 445 Ser Leu 460 Gly Leu Cys His 430 Pro Thr Ser Gin Ile 415 Ile Leu Glu Glu Ala 400 Ser Thr Leu Thr ~ro eu Ser Asn Ala 75 Arg Tyr Pro Ser Leu Leu Asp Asp Cys Val Thr Lys Tyr Phe Ser Leu Met Glu Ala Gly Met Lys Gin Ser Let.

lic Val Asn Se2 125 Asp Val Ph4 140 Leu Leu Lys Ala Arg Thr Thr Ser Leu Lys Lys Glu LGin Gly Arg Leu SGin Gin WO 98/46622 PCT/US98/07996 Val 145 Cys Pro His Tyr Arg 225 Pro Arg Ser Tyr Ser 305 Asp Cys Met Thr Asi 38! G1l As Th Pr Al 46 Glu His I] Asn Leu AE Cys Thr TI 1i Lys Ala L 195 Gly Met L 210 Arg Gin T Asn Cys L Ser Arg L 2 Val Ser A 275 Ser Gly I 290 Ser Leu S Leu Glu I Leu Lys Trp Gin 355 SAla Phe 370 i Glu lle n Lys Leu p Ser Asp r Thr Lys 435 o Val Leu 450 .a Glu Thr le sp hr 830 eu eu hr eu leu 60 %sn jeu Ser Asp Asn 34C Prc ArC Pr Ly Ph 42 Se Me Se Ser Lys Gly Asn 150 Asp Thr Cys Lys 165 Ser Met Ser Asn Arg Gin Phe Phe 200 Phe Cys Ser Cys 215 Ile Val Pro Val 230 Ser Leu Gin Asp 245 Ala Asp Phe Phe Cys Leu Lys Glu 280 Ile Gly Thr Val 295 Val Ala Pro Trp 310 SCys Leu Lys Phe 325 Ala Ile Gin Ala Ala Pro Pro Va] 36C g Val Lys Asn Lyi 375 o Thr His Val Lei 390 s Ser Asn Val Se: 405 e Gly Lys Asp Gl 0 .r Met Ala Ala Pr 44 it Leu Thr Ala Le 455 ?r Asn Cys Leu Asp Al 155 Lys Tyr Arg Ser A: 170 Glu Val Cys Asn A: 185 Asp Lys Val Pro A 2 Arg Asp Ile Ala C 220 Cys Ser Tyr Glu G 235 Ser Cys Lys Thr A 250 Thr Asn Cys Gin P 265 Asn Tyr Ala Asp C 2 Met Thr Pro Asn 300 Cys Asp Cys Ser I 315 Leu Asn Phe Phe 330 SPhe Gly Asn Gly 345 SGin Thr Thr Thr s Pro Leu Gly Pro 380 Pro Pro Cys Ala 395 r Gly Ser Thr His 410 y Leu Ala Gly Ala 425 o Pro Ser Cys Ser 0 .u Ala Ala Leu Leu 460 la Ala Lys Ala 160 la Tyr Ile Thr 175 rg Arg Lys Cys 190 la Lys His Ser ys Thr Glu Arg lu Arg Glu Arg 240 sn Tyr Ile Cys 255 ro Glu Ser Arg 270 ys Leu Leu Ala Tyr Val Asp Ser Asn Ser Gly Asn 320 Lys Asp Asn Thr 335 Ser Asp Val Thr 350 Ala Thr Thr Thr 365 Ala Gly Ser Glu Asn Leu Gin Ala 400 Leu Cys Leu Ser 415 Ser Ser His Ile 430 Leu Ser Ser Leu 445 Ser Val Ser Leu WO 98/46622 PCT/US98/07996 94 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "TrnRl primer 1" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GCGGTACCAT GTTCCTAGCC ACTCTGTACT TCGC 34 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "TrnR1 primer 2" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID GCTCTAGACT ACGACGTTTC TGCCAACGAT ACAG 34 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Trn2 primer 1" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GCGGTACCAT GATCTTGGCA AACGTCTGC 2! WO 98/46622 PCT/US98/07996 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "TrnR2 primer 2" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GCTCTAGAGT CAGGCGGCTG TTCTTGTCTG CG 32 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 90 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "NTN Primer 1" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ \D NO:18: GCATATGCCG GGTGCTCGTC CGTGCGGCCT GCGTGCAACT GGAAGTTCGT GTTTCTGAAC TGGGTCTGGG TTACACTTCT GACGAAACTG T INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 87 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "NTN Primer 2" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GCTGACGCAG ACGACGCAGA CCCAGGTCGT AGATACGGAT AGCAGCTTCG CATGCACCAG CGCAGTAACG GAACAGAACA GTTTCGT 87 WO 98/46622 PCT/US98/07996 96 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 87 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "NTN Primer 3" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID CTGCGTCAGC GTCGTCGTGT TCGTCGTGAA CGTGCTCGTG CTCACCCGTG CTGCCGTCCG ACTGCTTACG AAGACGAAGT TTCTTTC 87 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 86 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "NTN Primer 4" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CGGATCCTTA AACGCAAGCG CATTCACGAG CAGACAGTTC CTGCAGAGTG TGGTAACGAG AGTGAACGTC CAGGAAAGAA ACTTCG 86 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGOLINKER A" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: TATGCACCAT CATCATCATC ATGACGACGA CGACAAGGC 3! WO 98/46622 PCT/US98/07996 97 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OLIGOLINKER B" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: TAGCCTTGTC GTCGTCGTCA TGATGATGAT GATGGTGCA 39 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "GDNF PRIMER" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CAGCATATGT CACCAGATAA ACAAGCGGCG GCACT INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "GDNF PRIMER" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID CAGGGATCCG GGTCAGATAC ATCCACACCG TTTAGC 3E WO 98/46622 PCT/US98/07996 98 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "MOUSE RET FORWARD PCR

PRIMER"

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: TGGCACACCT CTGCTCTATG INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "MOUSE RET REVERSE PCR

PRIMER"

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: TGTTCCCAGG AACTGTGGTC INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "TrnRl FORWARD PRIMER" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: GCACAGCTAC GGGATGCTCT TCTG 2' WO 98/46622 PCT/US98/07996 99 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "TrnRl REVERSE PRIMER" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GTAGTTGGGA GTCATGACTG TGCCAATC 28 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "TrnR2 FORWARD PRIMER" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID AGCCGACGGT GTGGCTCTGC TGG 23 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "TrnR2 REVERSE PRIMER" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: CCAGTGTCAT CACCACCTGC ACG 2:

Claims (22)

1. An isolated and purified polypeptide comprising a TGF-P related neurotrophic receptor 2 (TrnR2) polypeptide.

2. The isolated and purified polypeptide of claim 1 wherein the TrnR2 polypeptide has a human amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:7 or wherein the TrnR2 polypeptide has a mouse amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:8.

3. The isolated and purified polypeptide of claim 1 wherein said TrnR2 popypeptide is a soluble TrnR2 polypeptide or soluble TrnR2 fragment, wherein said fragment is biologically equivalent to TrnR2.

4. A composition comprising the isolated and purified polypeptide of claim 1 in a pharmaceutically acceptable carrier.

5. The composition of claim 4 further comprising a TGF-P Related Neurotrophic (TRN) growth factor.

6. An isolated and purified polypeptide that is a member of the TGF-p related neurotrophic receptor family (TrnR) comprising an amino acid sequence having at-least sequence identity with the TrnR2 polypeptide of claim 1. 9

7. The isolated and purified polypeptide of claim 6 wherein said TrnR family member is comprised of a conserved region amino acid sequence having at least sequence identity with SEQ ID NO:9 or at least sequence identity with SEQ ID NO:10 or at least sequence identity with SEQ ID NO:ll.

8. An isolated and purified polynucleotide comprising a nucleotide sequence encoding the TrnR2 polypeptide of claim 1.

9. The isolated and purified polynucleotide of claim 8 wherein the TrnR2 polypeptide has a human amino acid sequence selected from the group consisting of SEQ ID WO 98/46622 .PCT/US98/07996 101 NO:2, SEQ ID NO:3, and SEQ ID NO:7 or a mouse amino acid sequence selected from the group consisting of SEQ ID SEQ ID NO:6 and SEQ ID NO:8. The isolated and purified polynucleotide of claim 8 wherein the TrnR2 polynucleotide encodes a human or mouse soluble TrnR2 polypeptide or soluble TrnR2 fragment, wherein said fragment is biologically equivalent to TrnR2.

11. An isolated and purified polynucleotide comprising a nucleotide sequence which specifically hybridizes to a nucleotide sequence selected from the group consisting of: a coding sequence for an amino acid sequence of a human precursor TrnR2 as set forth in SEQ ID NO:2; a coding sequence for an amino acid sequence for a mature human TrnR2 as set forth in SEQ ID NO:3; a sequence complementary to the coding sequence 10 of and a sequence complementary to the coding sequence of wherein said polynucleotide binds to said nucleotide sequences of with higher affinity than to other TrnR family members.

12. A vector comprising a recombinant DNA molecule comprising expression regulatory elements operably linked to a nucleotide sequence encoding the TrnR2 polypeptide of claim 1.

13. A host cell transformed with the vector of claim 12.

14. A recombinant cell transformed with nucleotide sequences encoding for expression the TrnR2 polypeptide of claim 1 and a Ret protein tyrosine kinase receptor. WO 98/46622 PCT/US98/07996 102 c- A recombinant DNA method comprising: subcloning a DNA sequence encoding the TrnR2 polypeptide of claim 1 into an expression vector which comprises regulatory elements needed to express the DNA sequence; transforming a host cell with said expression vector; growing the host cell to produce a host cell culture; and harvesting the TrnR2 polypeptide and/or the DNA sequence from the host cell culture.

16. An isolated and purified antibody raised against the TrnR2 polypeptide of claim 1 or an epitope thereof wherein the antibody specifically binds to said TrnR2 polypeptide with a higher affinity than to any other TrnR family members.

17. A method for preventing or treating cellular degeneration or insufficiency comprising administering to a patient a therapeutically effective amount of the TrnR2 polypeptide of claim 1.

18. A method for detecting TrnR2 expression in a sample from a patient comprising detecting in the sample the presence of the TrnR2 polypeptide of claim 1 or detecting the presence of a mRNA encoding the TrnR2 5 polypeptide of claim 1 or a biologically inactive mutant thereof.

19. A method for promoting the growth and/or differentiation of a cell in a culture medium comprising administering to the cell the TrnR2 polypeptide of claim 1 and a TRN growth factor. An isolated and purified TrnR2 antisense polynucleotide comprising a nucleotide sequence which is complementary to and hybridizes to a naturally-occurring DNA or mRNA polynucleotide sequence encoding a TrnR2 polypeptide to prevent transcription and/or translation of the encoded TrnR2 polypeptide.

21. A method for treating a disease condition Smediated by the expression of a TrnR2 polypeptide in a WO 98/46622 PCT/US98/07996 103 cell comprising administering to the cell an inhibitory. effective amount of the isolated and purified antisense TrnR2 polynucleotide of claim

22. A method for treating a disease condition in a patient mediated by the expression of the TrnR2 polypeptide of claim 1 comprising administering to the patient a therapeutically effective amount of an isolated and purified anti-TrnR2 antibody which blocks Ret activation in the presence of endogenous NTN and/or GDNF.

23. A method for treating a disease condition in a patient mediated by the expression of a TRN growth factor comprising administering to the patient a therapeutically effective amount of the isolated and purified soluble TrnR2 polypeptide or soluble TrnR2 fragment of claim 3.

24. A method for screening compounds for TRN agonistic or antagonistic activity comprising incubating a test compound with the recombinaht cell of claim 14 in the presence or absence of a TRN and assaying for Ret protein tyrosine kinase activity. An isolated and purified polypeptide comprising a TGF-p related neurotrophic receptor 2 (TrnR2) polypeptide which comprises a human amino acid sequence as set forth in SEQ ID NO:7 or a mouse amino acid sequence as set forth in SEQ ID NO:8.

26. An isolated and purified polypeptide comprising a soluble TGF-p related neurotrophic receptor 2 (TrnR2) polypeptide which comprises a human amino acid sequence as set forth in amino acids 1-299 of SEQ ID NO:7 or a mouse amino acid sequence as set forth in amino acids 1- 299 of SEQ ID NO:8.